KR20160048999A - HUMAN iNKT CELL ACTIVATION USING GLYCOLIPIDS WITH ALTERED GLYCOSYL GROUPS - Google Patents

Abstract

(GSL) containing alpha -glucose (alpha -Glc) which preferentially stimulates human constant NKT (iNKT) cells. GSLs with alpha -glucose ([alpha] -Glc) which are stronger (weaker than in mice) but which exhibit expansion and / or activation of immune cells than those with alpha-galactose ([alpha] -Gal) . GSL containing alpha -glucose (alpha -Glc) and derivatives of alpha -Glc having F at position 4 and / or 6 are provided. A method for iNKT-independent induction of chemokines by GSL with a-Glc and derivatives thereof is described. Methods for immunostimulation in humans using GSL with a-Glc and derivatives thereof are provided.

Description

[0001] The present invention relates to human iNKT cell activation using a glycolipid having an altered glycosyl group. BACKGROUND OF THE INVENTION < RTI ID = 0.0 >

inventor:

Chi-Huey WONG, Alice L. YU, Tai-Na WU, Kun-Hsien LIN

The present invention relates generally to the field of immunotherapeutic agents. In particular, the disclosure of the present invention relates to a method of modulating invasive natural killer T (iNKT) cells in a human and transactivating downstream immune cells that stimulate cytokine and / or chemokine production to connect innate and acquired immunity Glycolipids and variants thereof.

Natural killer type T (NKT) cells are a unique group of T lymphocytes with great therapeutic potential in the treatment of diseases such as cancer and autoimmune disorders. Invariant natural killer T (iNKT) cells form a subset of regulatory T cells that are characterized by both innate and acquired immunity. In contrast to conventional T cells activated by peptides provided by MHC class I or II molecules, iNKT cells are associated with CD1d, a non-typical MHC I molecule expressed on antigen presenting cells (APC) A derivative is recognized.

α-GalCer (α-galactosylceramid), a glycolipid composed of α-linked galactose, sphingosine and acyl tail, was obtained by structural optimization of agelas spin isolated from marine sponge Agelas mauritianus . This has been shown to exhibit antitumor activity both in vitro and in vivo and has been shown to be the most potent ligand known to date for both mouse and human invariant natural killer T cells (iNKT cells).

Invariant NKT cells (iNKT cells) co-express the CD161 antigen (NK cell marker NK1.1 in mice and NKR-P1A in humans) with constant TCR- alpha chain (V? 14 / J? 18 in mice and V? 24 / J? 18 in humans) . Reference: (1) Lantz, O .; Bendelac, AJ Exp. Med. 1994, 180, 1097; (2) Dellabona, P .; Padovan, E .; Casorati, G .; Brockhaus, M .; Lanzavecchia, AJ Exp. Med. 1994, 180, 1171; (3) Makino, Y .; Kanno, R .; Ito, T .; Higashino, K .; Taniguchi, M. Int. Immunol. 1995, 7, 1157; And (4) Davodeau, F .; Peyrat, MA; Necker, A .; Dominici, R .; Blanchard, F .; Leget, C; Gaschet, J .; Costa, P .; Jacques, Y .; Godard, A .; Vie, H .; Poggi, A .; Romagne, F .; Bonneville, MJ Immunol. 1997, 158, 5603. They respond to a large amount of Th1 (e.g., IFN-y, IL-2) and Th2 (e.g., IL-4, IL- 6) Secrete cytokines. ^5-9 (5) Kawano, T .; Cui, J .; Koezuka, Y .; Toura, L; Kaneko, Y .; Motoki, K .; Ueno, H .; Nakagawa, R .; Sato, H .; Kondo, E .; Koseki, H .; Taniguchi, M. Science 1997, 278, 1626; (6) Yoshimoto, T .; Paul, WEJ Exp. Med. 1994, 179, 1285; (7) Arase, H .; Arase, N .; Nakagawa, K .; Good, RA; Onoe, K. Eur. J. Immunol. 1993, 23, 307; (8) Kawakami, K .; Yamamoto, N .; Kinjo, Y .; Miyagi, K .; Nakasone, C .; Uezu, K .; Kinjo, T .; Nakayama, T .; Taniguchi, M .; Saito, A. Eur. J. Immunol. 2003, 33, 3322; And (9) Nieuwenhuis, EE; Matsumoto, T .; Exley, M .; Schleipman, RA; Glickman, J .; Bailey, DT; Corazza, N .; Colgan, SP; Onderdonk, AB; Blumberg, RS Nat. Med. 2002, 8, 588. These secreted cytokines are then down-regulated by DCs, natural killer cells (NK), B cells, CD4 ⁺ T and CD8 ⁺ T cells, thereby bridging congenital and adaptive immunity The transfected immune cells can be transactivated. ^10-12 (10) Eberl, G .; MacDonald, HR Eur. J. Immunol. 2000, 30, 985; (11) Eberl, G .; Brawand, P .; MacDonald, HRJ Immunol. 2000, 165, 4305; And (12) Kitamura, H .; Ohta, A .; Sekimoto, M .; Sato, M .; Iwakabe, K .; Nakui, M .; Yahata, T .; Meng, PL; Koda, T .; Nishimura, S .; Kawano, T .; Taniguchi, M .; Nishimura, T. Cell. Immunol. 2000, 199, 37.

However, an inverse balance of Th1 and Th2 cytokines may limit the clinical application of alpha GalCer to treat various disorders. ^13-16 (13) Tahir, SM; Cheng, O .; Shaulov, A .; Koezuka, Y .; Bubley, GJ; Wilson, SB; Balk, SP; Exley, MAI Immunol. 2001, 167, 4046; (14) Dhodapkar, MV; Geller, MD; Chang, DH; Shimizu, K .; Fujii, S.; Dhodapkar, KM; Krasovsky, JI Exp. Med. 2003, 197, 1667; (15) Giaccone, G .; Punt, CJ; Ando, Y .; Ruijter, R .; Nishi, N .; Peters, M .; von Blomberg, BM; Scheper, RJ; van der Vliet, HJ; van den Eertwegh, AJ; Roelvink, M .; Beijnen, J .; Zwierzina, H .; Pinedo, HM Clin. Cancer Res. 2002, 8, 3702; And (16) Bricard, G .; Cesson, V .; Devevre, E .; Bouzourene, H .; Barbey, C; Rufer, N .; Im, JS; Alves, PM; Martinet, O .; Halkic, N .; Cerottini, J. C .; Romero, P .; Porcelli, SA; Macdonald, HR; Speiser, DEJ Immunol. 2009, 182, 5140.

SUMMARY OF THE INVENTION

Thus, many analogs have been designed to stimulate selective Th1 or Th2 cytokine responses in iNKT cells. Glycolipids with prominent Th1 are biased in both mice and humans, leading to excellent tumor protection in vivo. For example, glycoprotein lipase (GSL) with truncated sphingosin tail could drive the immune response in the Th2 direction and prevented autoimmune encephalitis. ¹⁷ Miyamoto, K .; Miyake, S .; Yamamura, T. Nature 2001, 413, 531. Meanwhile, GSL having a phenyl ring on the acyl chain induced Th1-biased cytokines in mice and humans and showed more potent anticancer activity against breast, lung and melanoma tumors in mice . ^18-19 (18) (Chang, YJ; Huang, JR; Tsai, Y. C; Hung, JT; Wu, D .; Fujio, M .; Wong, CH; Yu, AL Proc. Natl Acad Sci. USA 2007, 104, 10299 and (19) Wu, TN; Lin, KH; Chang, YJ; Huang, JR; Cheng, JY; Yu, AL; Wong, CH Proc. Natl Acad Sci USA 2011, 17275.

Investigation of the binary interaction between CD1d and glycolipids, as well as the three-way interactions between the iNKT TCR and the CD1d-glycolipid complex, elucidated the mechanism underlying these structure-activity relationships (SARs). As compared to αGalCer, phenyl GSL with the same glycosyl group exhibits stronger binary and triple interactions leading to a more Th1-biased response, and the biological response is the binding avidity of both mouse and human ). ^19-21 (19) Wu, TN; Lin, KH; Chang, YJ; Huang, JR; Cheng, JY; Yu, AL; Wong, CH Proc. Natl. Acad. Sci. USA 2011, 108, 17275; (20) Liang, PH; Imamura, M .; Li, X .; Wu, D .; Fujio, M .; Guy, RT; Wu, B. C; Tsuji, M .; Wong, CHJ Am. Chem. Soc. 2008, 130, 12348; And (21) Li, X .; Fujio, M .; Imamura, M .; Wu, D .; Vasan, S .; Wong, CH; Ho, DD; Tsuji, M. Proc. Natl. Acad. Sci. USA 2010, 107, 13010.

(GSL) containing an α-galactosyl group (αGal) and a phenyl ring on the acyl chain stimulates more invariably invariant NKT (iNKT) cells than α-galactosyl ceramide (αGalCer) . Their activity in mice and humans correlates with the binding of the three-way interactions between the iNKT TCR and the CD1d-CSL complex.

Invariant natural killer T (iNKT) cells are known to have significant immunoregulatory capacity due to their ability to produce vast amounts of effector cytokines. There is a need for improved glycoprotein quality that stimulates human constant NKT (iNKT) cells and regulates cytokine and chemokine production in humans.

Accordingly, the disclosure of the present invention is based on the finding that the glycocalypse gonad (GSL) with alpha -glucose (alpha -Glc) is more potent than the one with alpha -galactose (alpha-Gal) Lt; / RTI > and / or exhibit weaker) induction in mice and / or expansion and / or activation of immune cells. Changes in the 4'-OH direction on glycosyl groups induced different bioactivity in mice and humans. Changes in position 6 of the glycosyl group also showed various effects on the biological response. GSL having a-Glc and derivatives of a-Glc with F at positions 4 and / or 6 are disclosed herein. A method of inducing iNKT-independent chemokines by GSL with derivatives of? -Glc and? -Glc is disclosed. Immunostimulation methods are provided in humans using GSL with derivatives of? -Glc and? -Glc thereof.

The disclosure of the present invention provides a method for enhancing the immunogenicity of an antigen in a subject in need thereof, comprising the simultaneous administration with an adjuvant composition comprising the GSL of formula (I) or the combined administration of said antigen as a co-formulation ≪ / RTI >

According to the present invention, the use of GSL as an immunosuppressant induces the enhancement and / or expansion of the duration of the protective immunity induced by the antigen, which is at least partly due to the promotion and / or extension of the antigen-specific Thl-type response do.

The GSL-α-Glc-containing adjuvants of the present invention may be co-administered with any antigen, particularly an antigen derived from an infectious agent or tumor. Preferably, adjuvants and antigens are most preferably administered simultaneously in a single dose form.

In a further aspect, the invention provides a prophylactic and / or therapeutic method for treating a disease in a subject, comprising administering to the subject an adjuvant composition comprising GSL, the immunoprotective antigen. As specified herein, the methods may be useful for preventing and / or treating various infectious or neoplastic diseases.

In conjunction with the methods of the present invention, there are provided pharmaceutical and vaccine compositions comprising an optionally effective pharmaceutically acceptable carrier or excipient, as well as an immunologically effective amount of an antigen and an immunologically effective amount of an adjuvant selected from GSL within formula 1 .

Thus, in one aspect, the disclosure relates to a structural and functional sample of an immunoadjuvant compound of formula I, or a pharmaceutically acceptable salt thereof:

(I)

In this formula,

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , n and m are as described herein.

In some embodiments, R < ⁴ > is a compound of formula II:

≪ RTI ID = 0.0 &

In this formula,

i and R < ⁶ > are as described herein.

In some embodiments, R < ⁴ > is a compound of formula III:

(III)

In this formula,

j, k, R < ⁷ > and R < ⁸ > are as described herein.

Aspects of the disclosure also relate to a pharmaceutical composition comprising (i) a compound described herein in an amount sufficient to stimulate an immune response when administered to a subject comprising a human, and (ii) a medicament comprising a pharmaceutically acceptable excipient ≪ / RTI >

In some embodiments, the pharmaceutical composition comprises an antigen and a vaccine adjuvant. In certain embodiments, the antigen is a tumor antigen.

In some embodiments, the pharmaceutical composition comprises an anti-cancer therapeutic agent.

In some embodiments of the pharmaceutical composition, R ⁴ in the compound is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, wherein said compound is capable of inhibiting Th1 cytokines in a human with a minimal associated increase in Th2 cytokines .

Aspects of the present invention are directed to methods for stimulating an immune response in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition as described herein.

In some aspects, the compound is administered in an amount that is capable of elevating invariant natural killer T (iNKT) cells in humans.

In some aspects, administration of the compound increases cytokine and / or chemokine production in humans. In some embodiments, the cytokine production is sufficient to transactivate downstream immune cells. In some embodiments, the downstream immune cell comprises at least one of dendritic cells (DC), natural killer cells (NK), B cells, CD4 ⁺ T and CD8 ⁺ T cells.

In some aspects, cytokines include Thl cytokines. In some embodiments, Thl cytokines are selected from interferon-gamma (IFN-y), GM-CSF, TNFa, interleukin 2, interleukin 12 and interleukin 10.

In some aspects, the chemokine is selected from RANTES, MIP-1 alpha, KC, MCP-I, IP-10, and MIG.

In some aspects, administration of the composition has an anti-cancer effect. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver tumors, leukemia, solid tumors and carcinomas.

In some embodiments, R < ⁴ > is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, wherein the increase in Th1 cytokines in a human exceeds any increase in Th2 cytokines.

Aspects of the present invention are directed to a method for raising the production of invariant natural killer T (iNKT) cells in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition, wherein , The composition comprises a compound described herein. In some embodiments, the elevation of the level of iNKT is greater when compared to an increase resulting from the administration of equivalent amounts of glycated analogs, including alpha-galactose (alphaGal) as a glycosyl head group.

Aspects of the present invention are directed to a method for stimulating production of cytokines and / or chemokines in a human subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of a composition, Lt; RTI ID = 0.0 > cytokine / chemokine < / RTI >

In some aspects, cytokine production is sufficient to transactivate downstream immune cells. In some embodiments, the downstream immune cell comprises at least one of dendritic cells (DC), natural killer cells (NK), B cells, CD4 ⁺ T and CD8 ⁺ T cells.

In some aspects, the cytokine comprises a Th1 cytokine. In some embodiments, the cytokine is selected from interferon-gamma (TFN-y), GM-CSF, TNFa, interleukin 2, interleukin 12 and interleukin 10.

In some aspects, the chemokine is selected from RANTES, MIP-1 alpha, KC, MCP-I, IP-10, and MIG.

These and other aspects will be apparent from the following description of the preferred embodiments taken in conjunction with the following drawings, but the variations and variations herein may be affected without departing from the spirit and scope of the novel concept described herein.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are included to further illustrate certain aspects of the disclosure of the present invention, which form a part hereof, and which, together with a detailed description of the specific embodiments provided herein, It can be well understood.
Figure 1 shows: the structure of the glycolipid with alpha Gal or alpha Glc. 7DW8-5-Man is the only compound with αMan.
Figure 2 shows: Glycoprotein analogs with alpha Gal are stronger immunomodulators than those with alpha Glc in mice, but are less immunomodulatory in humans. Glycolipid (1 [mu] g / mouse) or vehicle designated in B6 wild type and J [alpha] 18 knockout mice (n = 4) was intravenously injected. Serum collected at 2 hours and 18 hours after injection was analyzed for secretion of cytokines such as IFN-y (2A) and IL-4 (2B). IFN-y and IL-4 peaked at 18 hours and 2 hours after injection, respectively. (2C), B6 WT mice treated with the specified glycolipid (1㎍ / mouse) or vehicle (1% DMSO in PBS) are sacrificed 72 hours after the second injection of these spleen cells were FACS analysis for the determination of total CD3 ⁺ cells . (2D) human pure Vα24 + iNKT cells were cultured on day 2 with 100 ng / ml of designated glycolipid or autologous immature CD14 + DC pulsed with DMSO. After antigen removal on day 3, human iNKT cells were cultured in the presence of IL-2. The number of expanded iNKT cells was counted on the 9th day using the Guava ViaCount reagent. The Student's t test was selected to analyze the difference between 7DW8-5 and 7DW8-5-Glc and was p <0.001. Analysis was performed on two different donors with similar trends.
Figure 3 shows the following: The three-way interaction of CD1d-glycolipids with iNKT cells. (3A) DN3A4-1.2 Vαl4 + iNKT hybridoma cells and (3B) 7DW8-5-expanded Vα24 + iNKT cells were incubated with various concentrations of the designated dimeric mCD1d-glycolipids and hCD1d-glycolipid complexes at 4 ° C for 30 minutes, Respectively. Levels of bound complexes at the indicated concentrations were detected by anti-mIgGl secondary antibody and analyzed by flow cytometry. The relationship between% binding and concentration of CD1ddi-glycolipid complexes was plotted in mouse (3A) and human (3B). The KD values in mouse (3C) and human (3D) were calculated from the Scatchard transformation of plots (3A) and (3B), respectively. Analysis was performed twice.
Figure 4 shows: mCD1d vs. hCD1d swapping analysis. (4A) rat DN3A4-1.2 Val4 + iNKT hybridoma cells or (4B) C1-expanded Vα24 + iNKT cells were transfected into mCD1d (A20-CD1d cells) or hCDld (HeLa-CD1d cells) at 1, Lt; / RTI &gt; After 18 hours, the supernatant was collected and the IL-2 secretion was measured by ELISA analysis (4A) or using the Beadlyte® human cytokine kit and the Luminex® 200TM readout system (4B). Analysis was performed three times. 8-5 was the abbreviation for 7DW8-5.
Figure 5 shows the following: Computer modeling of the terrestrial complex of CDld-GSL-iNKT TCR. (5A) / (5B) hydrogen bonds in the CD1d-C1-iNKT TCR complex of mouse (5A) and human (5B). Formation of hydrogen bonds was noted in conserved residues including CD1d of iNKT TCR and human Asp80 (murine Asp80), human Thr154 (murine Thrl56), and human Asp151 (murine Asp 153) of human Gly96 (murine Gly96). In addition, mouse Asn30 and human Phe29 and Ser30 of iNKT TCR were key residues that formed H-bond interactions with 3'- and / or 4'-OH of Cl. The central 4'-OH of (5C) glucose can compensate for the loss of Phe29 interaction by interacting more strongly with the number of crystals captured by human iNKT TCR-Phe51 and hCDld-Trp153. (5D) aromatic interaction drives the acyl chain of C34 or C34-Glc to the lower position (near Cys 12) of the A 'channel in CD1d, resulting in subtle fluctuations in the orientation of the head group . (5E) Calculated free energy of the terrestrial complex using Autodock 4.2
Figure 6 shows the following: dose-dependent chemokine secretion induced by 7DW8-5-Glc. B6 wild-type mice were intravenously injected with 7DW8-5-Glc at 0.1 or 1 g / mouse. Serum collected at 2 hours and 18 hours after injection was analyzed for chemokine secretion such as IP-10 (6A), KC (6B), MCP-1 (6C), and MIP-1α (6D). These chemokines peaked at 2 hours after injection.
Figure 7 shows the following: iNKT-dependent production of cytokines and chemokines. B6 wild-type and Jα18 knockout mice were intravenously injected with the designated glycolipid (1 μg / mouse) or vehicle. Serum collected at 2 hours and 18 hours after the injection showed cytokines such as IL-2 (7A), IL-6 (7B), GM-CSF (7C) and TNF? Were analyzed for chemokines such as MIG (7F), KC (7G) and MCP-1 (7H). MIG alone was the peak at 18 hours after injection and the others peaked at 2 hours after injection.
Figure 8 shows: FACS analysis of WT mouse immune cells after designated glycolytic stimulation. B6 WT mice treated with the indicated glycolipid (1 μg / mouse) or vehicle (1% DMSO in PBS) were sacrificed at 72 hours post-injection and their spleen cells were subjected to FACS analysis. (8A) total spleen cells, (8B) total CD11Chi cells, (8C) CD11Chi / CD80 + cells, (8D) CD11Chi / CD86 + cells, (8E) CD4 + T cells and (8F) CD8 + T cells.
Figure 9 shows: FACS analysis of Jα18 KO mouse immune cells after designated glycolipid stimulation. B6 Jα18 KO mice treated with the indicated glycolipid (1 μg / mouse) or vehicle (% DMSO in PBS) were sacrificed at 72 hours post-injection and their spleen cells were subjected to FACS analysis. (9A) total splenocytes, (9B) total CD11Chi cells, (9C) CD11Chi / CD80 + cells, (9D) CD11Chi / CD86 + cells, (9E) CD4 + T cells and (9F) CD8 + T cells p < 0.05, as compared to D)
Figure 10 shows: the binding strength of the binary complex between mCD1d and the glycolipid. Different concentrations of mCD1d-glycolipid complexes coated on (10A, 10B) ELISA plates were incubated with a saturating amount of L363 conjugated with biotin followed by streptavidin-HRP detection and ELISA. The relationship between the OD value reflecting the (10A) L363 antibody binding and the concentration of the CD1ddi-glycolipid complex was plotted. (10B) The dissociation constant (KD) between the L363 antibody and the designated mCD1d-glycolipid complex was calculated as described in Materials and Methods. The relationship between the OD value reflecting the (1OC) L363 antibody binding and the concentration of glycolipids was plotted. The KD value of the (10D) binary complex was calculated from the linear regression analysis of the Sakacard transformation of the L363 antibody binding curve (1OC).
Figure 11 shows: CD1d dimer staining of in vivo C1-pulsed splenocytes. B6 WT splenocytes (n = 3) were stained with designated dimeric complexes conjugated with CD3, CD45R and RPE for 1 hour at 4 ° C after collection on day 3 after injection of C1 (1 μg / mouse). (11A) CD3 + / CD45R- cells were gated and analyzed for dimer staining. (11B) unloaded dimer was used as a control. (11C) mCD1d dimer loaded with 7DW8-5-Glc stained 17.1 + 0.8% of C1-pulsed splenocytes. (11D) Cloned 7DW8-5 of pulsed splenocytes 36.2 + 5.0% loaded mCD1d dimer.
Figure 12 shows the following: mCDld vs. hCDld swapping analysis. Cl-expanded Vα24 + iNKT cells were pulsed with the designated glycolipid provided by mCD1d (A20-CD1d cells) or hCD1d (HeLa-CD1d cells) at 1, 0.1 and 0.01 μg / ml. After 18 hours, the supernatant was collected for determination of IFN-y (12A) and IL-4 (12B) secretion. The ratio of IFN-y to (12C) IL-4 was calculated at different concentrations of glycolipids. The ratio of IFN-y to IL-4 of different gland origin was compared with the ratio from C1 at the concentration specified by the Student t test (*, p <0.05; **, p <0.01; ***, p < 0.001). Analysis was performed three times.

Natural killer T cells (NKT) represent a subset of T lymphocytes with unique properties, including the responsiveness to natural or synthetic glycolipids provided by CD1d and the expression of the constant T cell antigen receptor (TCR) alpha chain. NKT has been shown to functionally differentiate in the sense that they both share the properties of both natural killer cells and T cells and can rapidly produce both TH1- and TH2-type responses when stimulated with their ligands (innate immunity) Lt; RTI ID = 0.0 &gt; a T cells. &Lt; / RTI &gt; Activation of NKT can paradoxically induce inhibition or stimulation of the immune response. For example, the production of TH1 cytokines may be thought to promote cellular immunity along with antitumor, antiviral / anti-bacterial and adjuvant activities, while TH2 cytokine production is thought to calm autoimmune disease and promote antibody production . Because NKT plays a modulatory role in the immune system, they are attractive targets for immunotherapy.

As described above, the disclosure of the present invention is based on the fact that the glycocalypse gonad (GSL) with alpha -glucose (alpha -Glc) is more potent than the one with alpha -galactose Weak &lt; / RTI &gt; cytokines and chemokines and expansion and / or activation of immune cells. Thus, a composition comprising a method and exemplary GSL is provided herein.

Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology, microbiology, recombinant DNA and immunology within the skill of the art. This technique is fully described in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Culture of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. Eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lanes (Cold Spring Harbor Laboratory Press, 1988); and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986). In addition, methods of making and using immunosuppressants are described in U.S. Patent Nos. 7,488,491 and 7,928,077.

The term "lipid " as used herein refers to any lipophilic (lipophilic) molecule that participates in cellular signaling. The term "glycolipid " as used herein refers to a carbohydrate-attached lipid that acts as a marker for cell recognition.

The term "glycan" as used herein refers to polysaccharides or oligosaccharides. Glycans are also used herein to refer to carbohydrate moieties of the sugar conjugate, such as glycoproteins, glycolipids, glycopeptides, glycoproteom, peptidoglycan, lipopolysaccharide, or proteoglycan. Glycans generally consist solely of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically, glucan) composed of beta-1,4-linked D-glucose, and chitin is glycans composed of beta-1,4- linked N-acetyl-D-glucosamine to be. Glycans may be homologous or heteropolymers of monosaccharide residues and may be linear or branched. Glycans can be found to be attached to proteins, such as in glycoproteins and proteoglycans. These are generally found on the outer surface of the cell. O- and N-linked glycans are very common in eukaryotes but can also be found in prokaryotes, although less common. N-linked glycans were found to be attached to the R-group nitrogen (N) of asparagine in the sequence. The sequence is the Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except proline.

The term "glycoprotein " as used herein refers to a protein that is covalently modified with glycan (s). There are four types of glycoproteins: 1) N-linked glycoprotein, 2) O-linked glycoprotein (mucin), 3) glucose aminoglycan (GAG, also called proteoglycan), 4) GPI - Anchored. Most glycoproteins have structural micro-heterogeneity (multiple different glycan structures attached within the same glycosylation site) and structural macro-heterogeneity (multiple site and glycan attachment types).

The term "analog" as used herein refers to a compound, e. G., A drug, the structure of which is related to the structure of another compound, but its chemical and biological properties may be very different.

The term "antigen" as used herein is defined as any substance capable of causing an immune response.

The term "pathogen" as used herein is a biological agent that causes a disease or disorder in its host. The body is in the form of the human immune system and contains many natural defenses against some of the common pathogens (e.

The term "immunogen " as used herein refers to an antigen or substance capable of inducing the production of an antigen, such as a DNA vaccine.

The term "immunogenicity " as used herein refers to the ability of an immunogen, antigen or vaccine to stimulate an immune response.

The term "immunotherapeutic agent " as used herein refers to a series of therapeutic strategies based on the concept of modulating the immune system to achieve prophylactic and / or therapeutic goals.

Other definitions

The terms " treating "and" treating ", as used herein, refer to the reduction and / or elimination of the severity and / or incidence of symptoms, the elimination of symptoms and / or underlying causes and / Is used to refer to the administration of the agent or formulation to a clinically symptomatic individual suffering from a side effect, disorder or disease. The terms "preventing" and "prophylaxis" refer to the administration of a preparation or composition to a clinically asymptomatic subject that is susceptible to a particular adverse event, disorder or disease and thus prevents the occurrence of symptoms and / do. Unless otherwise indicated, obviously or implicitly, the term "treating" (or "treating") is intended to also encompass prevention if it is used without reference to possible prevention.

&Quot; Optional "or" optionally present ", as in "optional substituent" or "optionally present additive ", may or may not be present in the subsequently described components (e.g., substituents or additives) Quot; means that the component is present and that it is not present.

"Pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, for example, the substance can be used to induce or interact with any other undesirable biological effect Or may be incorporated into the formulation of the present invention. However, when the term "pharmaceutically acceptable" is used to refer to a pharmaceutical excipient, it means that the excipient meets the required criteria of toxicological and manufacturing testing and / Is included in the composition guide. As described in further detail below, "pharmacologically active" (or simply "active") as in a "pharmacologically active" derivative or analog is a derivative having the same type of pharmacological activity as the parent agent or Analogs.

The term "immunogen" as used herein refers to an antigen, or a substance capable of inducing the production of an antigen, such as a DNA vaccine.

The term "immunogenicity " as used herein refers to the ability of an immunogen, antigen or vaccine to stimulate an immune response.

The term "immunotherapy" as used herein refers to a series of therapeutic strategies based on the concept of modulating the immune system to achieve prophylactic and / or therapeutic goals.

The term "cytokine" as used herein refers to a molecule that modulates the intensity and persistence of an immune response by affecting the immune cell differentiation process, typically involving a change in gene expression that causes the precursor cells to become a unique specialized cell type Refers to any of a number of small secreted proteins. Cytokines are variously referred to as lymphocytes, interleukins and chemokines based on their putative functions, secretory cells or action targets. For example, some conventional interleukins include IL-12, IL-18, IL-2, IFN-y, TNF, IL-4, IL-10, IL-13, IL-21 and TGF- It is not limited. "Cytokine" is a generic term for a group of proteins released by one cell population acting on another cell population as an intracellular mediator. Examples of such cytokines are lymphokines, monokines and conventional polypeptide hormones. (IL-1, IL-2, IL-4, IL-10, IL-12), colony stimulating factor (CSF), thrombopoietin (PTH), erythropoietin (EPO), leukemia inhibitory factor (LIF), kit-ligand, growth hormone (GH), insulin growth factor (IGF), parathyroid hormone, thyroxine, insulin, ricassin, follicle stimulating hormone ), Thyroid stimulating hormone (TSH), luteinizing hormone (LH), hematopoietic growth factor, liver growth factor, fibroblast growth factor (FGF), prolactin, placental lactogen, tumor necrosis factor (TNF) (VEGF), integrin, nerve growth factor (NGF), platelet growth factor, transforming growth factor (TGF), bone morphogenetic ligand, etc. .

The term "chemokine" as used herein refers to any of a variety of small chemotactic cytokines released at the site of infection providing a means for migration and activation of lymphocytes. Chemokines cause white blood cells to become infected. Chemokines have conserved cysteine residues that allow them to be assigned to four groups. These groups, along with representative chemokines, are the C-C chemokines (RANTES, MCP-1, MIP-1α, and MIP-1β), C-X-C chemokine (IL-8), C chemokine (lymphotactin), and CXXXC chemokine (Pactalkine).

The term "epitope " as used herein is defined as a portion of an antigen molecule that contacts an antigen-binding site of an antibody or T cell receptor.

To further illustrate the differential binding of the 3-membered complexes in mice and humans, computer modeling was performed using x-rays of the mouse and human CD1d-αGalCer-iNKT TCR complex (PDB access codes 3HUJ, 3QUX, 3QUY, 3QUZ, and 3HE6) Structure. ^27-29 (27) Borg, NA; Wun, KS; Kjer-Nielsen, L .; Wilce, MC; Pellicci, DG; Koh, R .; Besra, GS; Bharadwaj, M .; Godfrey, DI; McCluskey, J .; Rossjohn, J. Nature 2007, 448, 44; (28) Pellicci, DG; Patel, O .; Kjer-Nielsen, L .; Pang, SS; Sullivan, LC; Kyparissoudis, K .; Brooks, AG; Reid, HH; Gras, S.; Lucet, IS; Koh, R .; Smyth, MJ; Mallevaey, T .; Matsuda, JL; Gapin, L .; McCluskey, J .; Godfrey, DI; Rossjohn, J. Immunity 2009, 31, 47; And (29) Aspeslagh, S .; Li, Y .; Yu, ED; Pauwels, N .; Trappeniers, M .; Girardi, E .; Decruy, T .; Van Beneden, K .; Venken, K .; Drennan, M .; Leybaert, L .; Wang, J .; Franck, RW; Van Calenbergh, S .; Zajonc, DM; Elewaut, D. EMBO J. 2011, 30, 2294.

The term "vaccine " as used herein refers to all disease-causing organisms (killed or attenuated) or proteins, peptides or polysaccharides used to confer immunity to an organism-induced disease RTI ID = 0.0 &gt; a &lt; / RTI &gt; The vaccine preparation may be native or synthesized or derived from recombinant DNA technology.

The term "immunogenic adjuvant " as used herein refers to a substance that is used in conjunction with an immunogenic source that enhances or modifies an immune response to an immunogenic source. Specifically, the terms "adjuvant" and "adjuvant" are used interchangeably herein and when administered alone to a host may be non-immunogenic, but when administered simultaneously with the antigen, &Lt; / RTI &gt; The enhancement of the adjuvant-mediated immune response and / or the extension of the duration of the immune response may be assessed by any method known in the art and include, without limitation, one or more of the following: (i) An increase in the number of antibodies produced in response to immunization with adjuvant / antigen combination compared to those produced in response to immunization with a single antigen; (ii) an increase in the number of T cells recognizing an antigen or adjuvant; And (iii) increased levels of one or more type I cytokines.

Exemplary adjuvants of the present invention include those compounds represented by formula (I).

Preferably, the exemplary adjuvants of the invention are pharmaceutically acceptable for use in humans.

The adjuvants of the present invention may be administered as part of a pharmaceutical or vaccine composition comprising an antigen or as a separate formulation administered with a second composition containing the antigen. In any of these compositions, glycocalgia (GSL) with alpha -glucose ([alpha] -Glc) may be combined with other adjuvants and / or excipients / carriers. These other adjuvants include oil-emulsions and emulsifier-based adjuvants, such as complete pruning adjuvants, incomplete pruning adjuvants, MF59 or SAF; Mineral gels such as aluminum hydroxide, aluminum phosphate or calcium phosphate; (CT), pertussis toxin, Escherichia coli heat-labile toxin (LT), mutant toxins (e. G., LTK563 or LTR72), basil calmette- Ergin (BCG), Corynebacterium parvum, DNA CpG motif, muramyldipeptide, or monophosphoryl lipid A; Microparticle adjuvants such as the immunostimulatory complex (ISCOM), liposomes, biodegradable microspheres, or saponins (e.g., QS-21); Cytokines, such as IFN-y, IL-2, IL-12 or GM-CSF; Synthesis auxiliaries such as nonionic block copolymers, muramylmethyl peptide analogs (e.g., N-acetyl-muramyl-L-tryonyl-D-isoglutamine [thr-MDP] Alanyl-2- [1'-2'-dipalmitoyl-sn-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl- Glycero-3-hydroxyphosphoryloxy] -ethylamine), polyphosphazene, or synthetic polynucleotides, and surface active substances such as resole lecithin, pluronic polyols, polyanions, peptides, hydrocarbon emulsions, Or keyhole limpet hemocyanin (KLH). Preferably, these additional adjuvants are also pharmaceutically acceptable for use in humans.

Within the meaning of the present invention, the term "co-administration or simultaneous administration" is used to refer to the administration of an antigen and an adjuvant simultaneously or sequentially in one composition, either simultaneously or in different compositions. However, for continuous administration to be considered "cavities ", the antigens and adjuvants must be administered separately at time intervals at which the adjuvant still enhances the immune response to the antigen. For example, if the antigen is a polypeptide, the antigen and adjuvant are administered on the same day, preferably within one hour of each other and most preferably simultaneously. However, when the nucleic acid is delivered to the subject and the polypeptide antigen is expressed in the cells of the subject, the adjuvant is administered within 24 hours, preferably within 6 hours, of the nucleic acid administration.

The term "immunogenicity " as used herein means that the agent can induce a humoral or cellular immune response, and preferably both. Immunogenic entities are also antigenic. An immunogenic composition is a composition that, when administered to an animal having an immune system, causes a humoral or cellular immune response, or both.

The term "antigen" refers to any agent which is recognized by the immune system of the host and which is capable of causing an immune response when introduced into a host, animal or human having an immune system (either directly or by expression, For example, proteins, peptides, polysaccharides, glycoproteins, glycolipids, nucleic acids or combinations thereof. As defined herein, the antigen-induced immune response may be humoral or cell-mediated or both. An agent is referred to as being "antigenic" when it is capable of specifically interacting with an antigenic molecule of an immune system such as an immunoglobulin (antibody) or T cell antigen receptor (TCR). Within the meaning of the present invention, the antigen is preferably a "surface antigen," a surface antigen that is naturally expressed on the surface of a pathogen or on the surface of an infected cell or on the surface of a tumor cell. The antigen-presenting molecule need not be an immunogenicity itself, that is, immunogenic capable of causing an immune response, without an adjuvant or carrier. The term " antigen specific "as used herein refers to the nature of the cell population that causes the supply of a particular antigen or fragment of said antigen to cause specific cell characteristics.

The term " epitope "or" antigenic determinant "refers to any part of an antigen that is recognized by B cells or T cells or both. Preferably, the interaction of said epitope with the antigen recognition site of an immunoglobulin (antibody) or T cell antigen receptor (TCR) results in induction of an antigen-specific immune response. T cells recognize proteins only if they are provided in a complex called "major histocompatibility complex (MHC)" that is cleaved into smaller peptides and located on another cell surface. There are two classes of MHC complex-I classes and II classes, each class consisting of many different alleles. The class I MHC complexes are found on virtually all cells and provide peptides that originate from proteins produced within the cell. Thus, a class I MHC complex is useful for killing cells infected by viruses or cells that become cancerous as a result of the expression of a carcinogenic gene. T cells with a protein called CD8 on their surface specifically bind to MHC / peptide complexes of the I class via T cell receptors (TCRs). This leads to cytolytic effector activity. The class II MHC complexes are used only to provide peptides that originate from circulating pathogens found only on antigen-presenting cells (APCs) and intracellularly transferred by APCs. T cells with a protein called CD4 bind to a class II MHC / peptide complex through TCR. This leads to the synthesis of specific cytokines that stimulate the immune response. To be effectively recognized by the immune system through the provision of class I MHC, the antigenic polypeptide must contain an epitope of at least about 8-10 amino acids, and in order to be effectively recognized by the immune system through the provision of class II MHC, antigenic poly The peptide must contain an epitope of at least about 13-25 amino acids. See Fundamental Immunology, 3rd Edition, W. E. Paul ed., 1999, Lippincott-Raven Publ.

The term "species-specific" antigen refers to an antigen present in or derived from a particular species. Thus, the term "malaria-derived" or "malaria-specific" antigen refers to a natural (e.g., irradiated sporozoite or synthetic (e.g., chemically- Or recombinantly synthesized polypeptides) antigens, which include Plasmodium (the plasmids include, but are not limited to, P. falciparum , P. vivax , p. malaria (P. malariae), P, O ballet (P. ovale), blood ray Kane nowi (P. reichenowi), blood, when no ulre (P. knowlesi), blood Sino drive (P. cynomolgi), P . bra Celia num (P. brasilianum), blood from Eli Io (P. yoelii), blood Berg Hay any one of proteins constituting the (P. berghei), or blood, a woody cover (P. chabaudi)) (B-cells and / or T-cells) and comprises at least 5 to 10 amino acid residues. The antigen A preferred protoplastic protein for sex is the circumsporozoite (CS) protein, but other proteins, such as thrombospondin-related adhesins (also called sporozoite surface protein 2 (SSP2) (TRAP), LSA I, hsp70, SALSA, STARP, Hep17, MSA, RAP-1, RAP-2 and the like can be used.

The term "vaccine" refers to a composition (e. G., A protein or vector such as an adenovirus vector, a sindbis virus vector or a poxvirus vector) that can be used to induce protective immunity in a recipient. It should be noted that the vaccine of the present invention can induce immunity in some of the immunized population, since some individuals may be resistant or may fail to increase the protective immune response or in some cases increase any immune response. Such inability may be determined by the genetic background or immunodeficiency conditions of the individual (acquired or congenital) or immunosuppression (e.g., treatment with chemotherapy or, for example, immunosuppressive drugs to prevent organ rejection or to inhibit autoimmune disease Due to the use of &lt; / RTI &gt; Vaccine efficacy can be established in animal models.

The term "DNA vaccine" is a common term in the art and is used herein to refer to a vaccine delivered by a recombinant vector. Another term and more technical term used herein is the term "vector vaccine" (some potential vectors, such as retroviruses and lentiviruses are RNA viruses and, in some cases, Is transmitted). In general, the vector is administered in vivo, but in vitro transduction of appropriate antigen-presenting cells such as dendritic cells (DCs), and administration of cells transduced in vivo is also contemplated.

The term "treating" as used herein means alleviating or alleviating at least one symptom of a disease in a subject. In the sense of the present invention, the term "treating" can also mean delaying the time between onset of infection, i.e., clinical signs of infection and disease. Preferably, the disease is selected from the group consisting of infectious diseases (e. G., Viral, bacterial, parasitic or fungal) or malignant tumors (e. G. Solid tumors or hematologic tumors such as sarcoma, carcinoma, glioma, , Breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia, melanoma, etc.).

As used herein, the term "protect" means suitably preventing or treating a disease or a continuing disease or both in a subject. Within the meaning of the present invention, the disease is selected from the group consisting of infections (e.g., viral, bacterial, parasitic or fungal) and / or malignant tumors (e.g. solid tumors or hematologic tumors such as sarcomas, carcinomas, Breast cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia, melanoma, etc.). For example, in accordance with the present invention, the therapeutic administration of a tumor-specific antigen in combination with an adjuvant comprising an exemplary formulation of Formula 1 may result in a delayed tumor growth and metastasis or even an antitumor immune response leading to tumor regression .

The term "protective immunity" refers to the inactivation and / or reduction in loading of the antigen, which prevents or slows the onset of the disease at repeated exposures to the same or related antigen, and long lasting immunity (e.g., (Active / acquired or passive / congenital, or both) in the host animal, leading to an acquired response through the immune system. "Protective immune response" means, for example, the removal of humoral (antibody) immunity or cellular immunity or both to reduce or reduce the loading of pathogens or infected cells, or to reduce tumor load in immunized (vaccinated) . Within the meaning of the present invention, protective immunity may be partial.

The immune system is divided into two general systems, the "innate" or "natural" immune system, and the "acquired" or "adaptive" immune system. The congenital immune system may initially allow the infection to be under control, allowing time for the adaptive immune system to develop an appropriate response. Recent studies have suggested that various components of the innate immune system induce and potentiate components of the adaptive immune system, including antigen-specific B and T lymphocytes (Fearon and Locksley, supra; Kos, 1998, Immunol. Res , 17: 303; Romagnani, 1992, Immunol. Today, 13: 379; Banchereau and Steinman, 1988, Nature, 392: 245).

The term "innate immunity" or "natural immunity" refers to a congenital immune response unaffected by prior contact with an antigen. Cells of the innate immune system, including macrophages and dendritic cells (DCs), absorb foreign antigens through pattern-recognition receptors, form MHC molecules of Class I and Class II with peptide fragments of these antigens and form pure CD8 ⁺ and CD4 ⁺ T cells (Banchereau and Steinman, supra; Holmskov et al., 1994, Immunol. Today, 15: 67; Ulevitch and Tobias, 1995, Annu. Rev. Immunol., 13: 437). Expert antigen-presenting cells (APCs) communicate with these T cells and induce differentiation of pure CD4 ⁺ T cells into T-helper 1 (Thl) or T-helper 2 (Th2) lymphocytes, (Abbott et al., 1996, Nature, 383: 787), which mediate the transcriptional regulation of endothelial cells (Trinchieri, 1995, Annu. Rev. Immunol., 13: 251; Howard and O'Garra, 1992, Immunol. Today, 13: ; Okamura et al., 1998, Adv. Immunol., 70: 281; Mosmann and Sad, 1996, Immunol. Today, 17: 138; O'Garra, 1998, Immunity, 8: 275).

The term " acquired immunity "or" adaptive immunity ", as used herein, refers to an amount of active or passive, humoral, and / or immune response that is established during the life of an animal and that is specific for an induced antigen and that is promoted by an enhanced response to repeated contact with the antigen Or cellular immunity. The key feature of the T lymphocytes of the adaptive immune system is then the ability to detect small concentrations of the pathogen-derived peptides provided by MHC molecules on the cell surface.

The term "enhancing the immune response " as used herein means enhancing or extending the immune response, or both. The term "enhancing immunity" when referring to the nature of an antigen (e. G., Adjuvant) refers to the ability to enhance the immunogenicity of the antigen or to extend the duration of the immune response to the antigen, Both are mentioned.

The term "enhancing the immune response " within the meaning of the present invention refers to a property or process that increases the magnitude and / or efficiency of the immune response to a given antigen, and the immunoreactivity refers to humoral or cellular immunity or Both are. The immune response is enhanced when any measurable parameter of antigen-specific immunoreactivity (e. G., Antibody titer, T cell production) is increased by at least 2-fold, preferably 10-fold, most preferably 30-fold .

The term "therapeutically effective amount" as applied to a dose or amount refers to the amount of a compound or pharmaceutical composition or vaccine sufficient to induce the desired activity upon administration to a mammal in need thereof. The term "therapeutically effective amount / effective dose" as used herein with respect to adjuvants and antigen-containing compositions is used interchangeably with the term " immunologically effective amount / effective dose " or an adjuvant comprising a glycoprotein lipase (GSL) having an alpha-Glc (alpha-Glc)) or an amount / dose of a pharmaceutical composition or vaccine sufficient to produce an effective immune response upon administration to the mammal.

The term "pharmaceutically acceptable " used in connection with the composition of the present invention refers to a molecular entity that is physiologically tolerable when administered to humans and typically does not produce unwanted reactions and other components of the composition. Preferably, the term "pharmaceutically acceptable ", as used herein, is intended to encompass all types of pharmaceutical preparations which are approved by the federal or state regulatory agency for use in mammals and, more particularly, It means that it is listed.

The term "carrier &quot;, as applied to the pharmaceutical or vaccine composition of the present invention, is intended to encompass an antigen comprising a glycoprotein lipase (GSL) having a compound (e.g., a-Glc) and / Adjuvant &quot;) is administered is referred to as a diluent, excipient or vehicle. The pharmaceutical carrier may be sterile liquids such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solutions, saline solutions, and aqueous dextrose and glycerol solutions are preferably used as carriers, especially for injectable solutions. Suitable pharmaceutical carriers include literature, it is described in [refer to "Remington's Pharmaceutical Sciences" by EW Martin, 18 th Edition].

The term "intrinsic antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of about 150,000 daltons, usually consisting of two identical light chain (L) chains and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of the different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intramolecular disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain having a variable domain (VL) at one end and a constant domain at the other end thereof; The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J Mol. Biol., 186: 651-663, 1985; Novotny and News, Proc. Natl. Acad Sci. USA, 82: 4592-4596, 1985).

The term "antibody" or "Ab" is used in its broadest sense and includes antibody fragments (e.g., Fab, F (ab ') 2, scFv and Fv) Monoclonal antibodies (including agonists and antagonist antibodies), antibody compositions with multivalent epitope specificity.

The term "CD1d" as used herein refers to a member of the CD1 (cluster of differentiation 1) family of glycoproteins expressed on the surface of various human antigen-presenting cells. The CD1d-provided lipid antigen activates natural killer T cells. CD1d has a deep antigen-binding groove to which the glycoprotein antigen binds. CD1d molecules expressed on dendritic cells can bind and provide glycoproteins.

The term "adaptive immune system " as used herein refers to processes that eliminate highly specialized systemic cells and pathogenic challenges. Cells in the adaptive immune system are a type of white blood cell called lymphocytes. B cells and T cells are the major types of lymphocytes.

The terms "T cell" and "Ts " as used herein refer to a leukocyte group known as a lymphocyte that plays a pivotal role in cell mediated immunity. T cells can be distinguished from other lymphocyte types, such as B cells and NK, by the presence of specific receptors on their cell surface, referred to as T cell receptors (TCRs). Several different subsets of T cells have been described and each has a unique function. Helper T (T _H ) cells are "brokers" of the adaptive immune system. Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or "help " the immune response. Depending on the accepted cytokine signal, these cells differentiate into one of T _H 1, T _H 2, T _H 17 or another subset that secretes different cytokines.

The term "antigen-presenting cell" (APC) as used herein refers to a cell that displays foreign antigens complexed with a major histocompatibility complex (MHC) on its surface. T-cells can recognize these complexes using their TCRs. APCs fall into two categories: professional or unprofessional. Dendritic cells (DCs) belong to the professional category and can provide antigens to T cells in the context of CD1. In an exemplary implementation, DCs used in the methods of the disclosure of the present invention may be any of a number of DC subsets that differentiate from a lymphocyte in one performance or from a myeloid bone marrow in another performance.

The term "pure cell " as used herein refers to a non-differentiated immune system cell, such as a CD4 T-cell, which has not yet been specified to recognize a specific pathogen.

The terms "natural killer cell" and "NK" as used herein refer to a class of lymphocyte cells activated by interferon to contribute to innate host defense against viruses and other intracellular pathogens.

The term "natural killer T cell" (NKT) as used herein refers to a subset of T cells that share a property / receptor with both conventional T and NK. Many of these cells recognize non-polymorphic CD1d molecules that are antigen-providing molecules that bind to self and foreign lipids and glycolipids. The TCR of NKT can recognize (chaperoned) glycolytic antigens provided by CD1d molecules. The major response of NKT is the secretion of rapid cytokines, including IL-4, IF-y and IL-10, after stimulation, thus affecting a variety of immune and pathogenic processes. The NKT may be a homogeneous group or a heterogeneous group. In one illustrative performance, the population is human, and mouse bone marrow and human liver T cells that express various TCRs and are capable of producing large amounts of IL-4 and IFN- [gamma], for example CD1d- Quot; non-invariant NKT ", which may include groups. A subset of the best known CD1d-dependent NKTs express an invariant TCR-alpha (TCR-alpha) chain. These are referred to as I-form or invariant NKT (iNKT). These cells are conserved between human (Vα24i NKT) and mouse (Vαl4i NKT) and are involved in many immunological processes.

As used herein, the term "cytokine" refers to a cytokine that modulates the intensity and duration of an immune response by affecting the immune cell differentiation process, which typically involves a change in gene expression, in which the precursor cells become a unique specialized cell type Refers to any of a number of small secreted proteins. Cytokines have been variously referred to as limpokines, interleukins and chemokines based on their putative functions, secretory cells or action targets. For example, some conventional interleukins include IL-12, IL-18, IL-2, IFN-y, TNF, IL-4, IL-10, IL-13, IL-21 and TGF- It is not limited.

The term "chemokine" as used herein refers to any of a variety of small chemotactic cytokines released at the site of infection providing a means for migration and activation of lymphocytes. Chemokines attract white blood cells to the site of infection. Chemokines have conserved cysteine residues that cause them to be assigned to four groups. Representative groups with chemokines are the C-C chemokines (RANTES, MCP-1, MTP-1α, and MIP-1β), C-X-C chemokine (IL-8), C chemokine (lymphotactin), and CXXXC chemokine (Pactalkine).

The term " T _H 2 type reaction "as used herein refers to a cytokine expression pattern that allows certain types of cytokines, interferons, and chemokines to be produced. Typical T _H 2 cytokines include, but are not limited to IL-4, IL-5, IL-6 and IL-10.

The term " T _H type response "as used herein refers to a pattern of cytokine expression that allows certain types of cytokines, interferons, and chemokines to be produced. Typical T _H 1 cytokines include, but are not limited to, IL-2, IFN-y, GM-CSF and TNF-β.

As used herein, the term "T _H 1 biased" as used refers to a T _H 1 cytokines and / or chemokines of the T _H 2 cytokine production and / or significantly increased immunogenic response than the chemokine production.

The term " antimicrobial "as used herein refers to a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi or viruses.

The term " toxoid " as used herein refers to a bacterial toxin whose toxicity is weakened or inhibited by chemical (formalin) or heat treatment, but other properties, typically immunogenicity, are maintained. Toxoids are used in vaccines because they induce an immune response to the native toxin or increase the response to another antigen. For example, tetanus toxoid is derived from tetanopathamine , which is produced by Clostridium tetani and causes tetanus. These tetanus toxoids have been used by many plasma centers in the United States for the development of plasma-rich vaccines.

The term "DNA vaccine " as used herein refers to a DNA construct that is introduced into a cell and subsequently decoded with a specific antigen protein.

The term "plasmid" as used herein refers to a replicable extrachromosomal circular DNA that can be used as a cloning vector.

The terms "microbe" and "microbe" as used herein refer to a microscopic organism that is too small to be visible to the human eye. Microorganisms are very diverse and include, but are not limited to, bacteria and fungi.

The term "adjuvant or immunoadjuvant " as used herein refers to a substance used in conjunction with an immunogen that enhances or modifies an immune response to an immunogen. In one example, the compounds / analogs of the disclosure of the present invention are used as immunological adjuvants to modify or enhance the vaccine effect by stimulating the immune system of a patient receiving the vaccine to respond more vigorously to the vaccine.

The term "alum adjuvant " as used herein refers to an aluminum salt with immunoadjuvant activity. Such agents adsorb and precipitate protein antigens in solution; The resulting precipitate improves vaccine immunogenicity by promoting slow release of the antigen from the vaccine depot formed at the site of vaccination.

The term " anti-tumor immunotherapeutic active agent " as used herein refers to exemplary compounds / analogs of the disclosure of the present invention that inhibit and / or reduce, and / or eliminate tumors.

The term "granulocyte-macrophage colony-stimulating factor" (GM-CSF) as used herein excludes leukocytes, particularly granulocytes (neutrophils, basophils and eosinophils), macrophages and platelets, Refers to a cytokine that acts as a colony-stimulating factor.

The term " antigen specific "as used herein refers to the nature of the cell population that causes the delivery of a particular antigen or fragment of said antigen to induce a particular cell proliferation.

The term "flow cell counting" or "FACS " as used herein means a technique for examining the physical and chemical properties of suspended particles or cells in a fluid stream through optical and electrical detection devices.

The amino acid residues in the peptide are then abbreviated as follows: phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic acid is Asp or D; Glutamic acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G. See Proteins: Structure and Molecular Properties by Creighton, T. E., W. H. Freeman & Co., New York 1983 for further description of amino acids.

Mammalian and mycobacterial lipids include human CD1a, CD1b, CD1c, and CD1d. It is known to be provided by? -galactosylceramides, and the lipids found in marine spongelized gelatin mauritian have been extensively studied as ligands for CD1d. In vitro stimulation of mouse spleen cells by alpha -GalCer induced stimulation of NKT and production of both IFN-D and IL-4, T _H 1 -type and T _H 2-type responses, respectively. Mouse studies have shown that cells can be rapidly activated by immature dendritic cells (iDC) containing alpha-GalCer and the activated iNKT can then induce complete maturation of DC.

The use of adjuvants comprising glycoside high-quality lipids (GSL) with? -glucose (? -Glc)

In one aspect, the present invention provides a method of treating an antigen in a mammal, comprising administering an antigen in association with an adjuvant composition comprising a glycoprotein lipase (GSL) having an alpha -glucose (alpha-Glc) Lt; RTI ID = 0.0 &gt; immunogenicity. &Lt; / RTI &gt; In accordance with the present invention, the use of glycosidic lipids (GSL) with alpha -glucose (alpha -Glc) induces an increase in the protective immunity and / or prolongation of the protective immunity induced by said antigen. For example, as described herein, a peptide corresponding to a T cell or B cell epitope of a tumor or viral antigen, or a glycoprotein having a-glucan (a-Glc) together with a DNA construct expressing these antigens, Co-administration of high-grade lipids (GSL) promotes an antigen-specific immune response.

The glycocalypse high-grade (GSL) -containing adjuvant with? -Glucose (? -Glc) of the present invention may be administered in conjunction with any antigen, particularly an antigen derived from an infectious agent or tumor.

In both mice and humans, immunostimulatory immunostimulatory effects can depend on the expression of CD1d molecules and are mediated by NKT cells. Indeed, the present invention demonstrates that the adjuvant activity is due at least in part to its ability to promote and / or prolong NKT-mediated antigen-specific Th1-type T cell responses and CD8 + T cell (or Tc) responses.

From an immunotherapeutic point of view, the glycoprotein lipase (GSL) with the a-glucose (? -Glc) activation of the NKT cell system appears to have a distinct advantage over other mechanisms for the following reasons: (a) &Lt; / RTI &gt; is very high and effective for a wide range of tumor cells or infected cells; (b) Activation of NKT cells by glycosidic lipid (GSL) with? -glucose (? -Glc) is dependent on the CD1d molecule as a whole in its entirety in individuals (Porcelli, Adv. Immunol. , 59: 1-98, 1995), indicating that glycoprotein-rich adjuvants with alpha -glucose (? -Glc) can be utilized by all patients regardless of MHC single phase form; (c) The antigen-donating function of DC and NKT activation in human patients can be assessed prior to immunotherapy by in vivo assay in mice using Val4 NKT cell status as indicator.

According to the present invention, adjuvants and antigens comprising glycosidic lipids (GSL) with alpha -glucose (alpha -Glc) of formula (I) can be administered as two separate formulations or as part of the same composition. If administered separately, the adjuvant and antigen may be administered subsequently or concurrently. As disclosed herein, simultaneous administration of an antigen with a glycocopherol (GSL) adjuvant with alpha -glucose ([alpha] -Glc) is preferred and generally allows the most efficient immune stimulation to be achieved.

Since the glycoprotein high-grade (GSL) adjuvant with? -Glucose (? -Glc) of the present invention exhibits its immunostimulatory activity in combination with a number of different antigens, therefore, both for prophylactic and therapeutic applications useful. Thus, in a further aspect, the present invention provides a method for the treatment and / or prophylaxis of a mammal, including co-administration of adjuvants and antigens comprising the glycocalypse gland (GSL) with? -Glucose (? The present invention provides prophylactic and / or therapeutic methods for treating diseases in animals. Such methods may be useful, for example, for treating various neoplastic diseases as well as for protecting against various infections and / or for treating infections.

The method of enhancing immunogenicity of the present invention may be used to treat a variety of conditions including, but not limited to, parasitic infections (e.g., infections caused by protoplasmic species), viral infections (e.g., influenza viruses, leukemia viruses, (E.g., papilloma virus, herpes virus, hepatitis virus, measles virus, poxvirus, mumps virus, cytomegalovirus [CMV], Epstein-Barr virus etc.), bacterial infections (for example, staphylococcus , (streptococcus), pneumophila Caucus (pneumococcus), Neisseria Kono Leah (Neisseria gonorrhea), borelri Ah (Borrelia), Pseudomonas (pseudomonas), infections caused by, etc.), and fungal infections (eg, Candida (candida), tricot piton (trichophyton), Pyrrhus of Epirus including Forum (ptyrosporum), infection) caused by an infection, but not limited to a compost I can get it.

The method of the present invention may also be used to treat a variety of conditions including but not limited to fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, choroidal carcinoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphovascular endothelial sarcoma, synovial sarcoma, mesothelioma, Leukemia, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, gland carcinoma, sebaceous adenocarcinoma, papillary adenocarcinoma, cystic carcinoma, thyroid carcinoma, bronchial carcinoma, pancreatic carcinoma, pancreatic carcinoma, pancreatic carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, Cancer of the uterine cervix, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, stromal cell carcinoma, hepatocellular carcinoma, hepatocellular carcinoma, hepatocellular carcinoma, choriocarcinoma carcinoma, choriocarcinoma carcinoma, Is useful for the treatment of various cancers including, but not limited to, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, craniopharyngioma, ependymoma, pineal gland, angioblastoma, auditory neoplasm, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma .

As further disclosed herein, the maximal efficacy of the immunogenicity-enhancing method of the present invention is obtained when the antigen, and the glycoside high-quality lipid (GSL) adjuvant with alpha -glucose (alpha -Glc) are administered concurrently.

The methods of the invention can be used in conjunction with other treatments. For example, chemotherapy and / or radiation therapy and / or radiation therapy using tumor-specific antigens, and glycoside high-purity (GSL) -containing adjuvants with the present alpha -glucose ([alpha] May be used in combination with IL-12 therapy. An anti-viral vaccine comprising a glycocopherol (GSL) -containing adjuvant with? -glucose (? -Glc) can be used in combination with IFN-a therapy.

Glycosidation High Glycemic (GSL) -containing pharmaceutical and vaccine compositions with? -glucose (? -Glc)

In conjunction with the method of the present invention, it is also possible to use an immunologically effective amount of an antigen, as well as optionally an additional immunostimulant, carrier or excipient (preferably both pharmaceutically acceptable), and a glycine with? -Glucose There is provided a pharmaceutical composition and vaccine composition comprising an immunologically effective amount of an adjuvant comprising a cosolventogenase (GSL). The antigens and adjuvants may be formulated as a single composition that can be administered simultaneously or sequentially, or as two separate compositions.

The adjuvants of the present invention comprise a compound belonging to the class of sphingoglycolipids, specifically glycosidic lipids (GSL) with alpha -glucose (alpha-Glc), which can be represented by formula (1)

Antigens used in the immunogenic (e.g., vaccine) compositions of the invention may be eukaryotic (e. G., Tumors, parasites, fungi), bacterial cells, virus cells, or any portion thereof Virus particles or virus components). If the substance to which the immunogenic response is to be directed is poorly antigenic, it may be further conjugated to a carrier molecule, such as albumin or hapten, using standard covalent bonding techniques, for example, with one of several commercially available reagent kits .

Examples of preferred tumor antigens of the invention include ErbB receptors, Melan A [MARTI], gp100, tyrosinase, TRP-1 / gp75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and non-small cell carcinoma); HPV EG and E7 proteins (in uterine cancer); Mucin [MUC-1] (in breast, pancreas, colon and prostate); Prostate-specific antigen [PSA] (in prostate cancer); MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-2, MAGE-4, MAGE-6, MAGE- Specific antibodies such as the shared tumor-specific antigens as LAGE-3 to 7, LAGE-1, NY-ESO-1 / LAGE-2, NA-88, GnTV, and TRP2-INT2. The previous list of antigens is intended to be exemplary since the antigen of interest can be delivered from any animal or human pathogen or tumor.

In certain embodiments, an antigen of the invention may be provided by a recombinant virus expressing said antigen. Preferably, the virus is selected from the group consisting of recombinant adenovirus, recombinant poxvirus and recombinant sindbis virus.

In the compositions described above, the antigen, and the glycoside high-grade lipid (GSL) adjuvant with alpha -glucose ([alpha] -Glc) are present in an immunologically effective amount. For each particular antigen, the optimal immunologically effective amount should be determined experimentally (taking into account the specific characteristics of the desired patient and / or type of treatment). Generally, the amount is an antigen ranging from 0.1 μg to 100 mg per kg of body weight. For the glycoprotein high-grade (GSL) adjuvant with? -Glucose (? -Glc) of the present invention, the optimal immunologically effective amount is preferably an adjuvant in the range of 10 to 100 μg per kg of body weight.

The present invention also provides a method for preparing a vaccine composition comprising one or more antigens and an adjuvant comprising glycosidic lipid (GSL) with alpha -glucose of formula (1) Includes the incorporation of adjuvants and antigens, and optionally one or more physiologically acceptable carriers and / or excipients and / or adjuvants.

Formulation and administration

The present invention also relates to a therapeutic agent of the present invention (an antigens as a single composition that can be administered simultaneously or subsequently, or as two separate compositions, and a glycoprotein quality (GSL) adjuvant with? -Glucose Wherein the formulation is suitable for administration to induce an antigen-specific protective immune response for the treatment and prevention of the infectious or neoplastic disease described above. The compositions of the present invention may be formulated in any conventional manner using one or more physiologically acceptable carriers or excipients. Thus, adjuvants comprising an antigen and / or a glycoprotein complex (GSL) with alpha -glucose of formula (1) can be administered orally, buccally, intranasally, intravaginally, intravaginally, rectally, Including, but not limited to, intraperitoneal, intramuscular, intraperitoneal, intravenous, subcutaneous routes, through exfoliation (scratching through the upper layers of the skin such as a bifurcated needle) Or by administration to a subject of the cell following administration by inhalation (lung) or insufflation (via the mouth or nose), or administration to an ex vivo antigen-presenting cell, or by administration of any other of the immunization May be formulated for administration by standard routes.

Preferably, the immunogenic formulations of the invention are administered parenterally, that is, intravenously (iv), subcutaneously (sc), intraperitoneal (ip), intramuscular (im), subcutaneous (sd), or transdermal For example, by bolus injection, continuous infusion, or by a gene gun (e.g., to administer a vector vaccine, e.g., leaking DNA or RNA, to a subject). The injectable formulations may be presented in unit dose form, for example, in ampules or in multi-dose containers, with an added preservative. The composition may take the form as an excipient, a suspending agent, a solvent or an emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e. G., A sterile pyrogenic member, prior to use.

The present invention also contemplates various mucosal vaccination strategies. Although the mucosa can be targeted by local delivery of the vaccine, a variety of strategies are used to deliver the immunogenic composition to the mucosa. For example, in certain embodiments, the immunogenic polypeptide or vector vaccine may be administered as a conjugate or chimeric fusion protein with, or in combination with, a chimeric toxin, such as cholera toxin B or cholera toxin A / B chimera Leibens and Holmgren, Giant Biol Stand 82: 215-27, 1994), which is described in Hansen et al., J. Immunol., 154: 4322-32, 1995; Jobling and Holmes, Infect Immun., 60: 4915-24, . In another embodiment, a mixture with a thermostable enterotoxin (LT) may be prepared for mucosal vaccination. Other mucosal immunization strategies include encapsulating immunogens in microcapsules (see, for example, US Pat. Nos. 5,075,109; 5,820,883; and 5,853,763), using immunoconjugated membrane carriers (see, for example, PCT WO 98/0558). The immunogenicity of an orally administered immunogen may be determined by using erythrocyte (rbc) or rbc ghost (see, for example, U.S. Patent No. 5,643,577), or a nephrotic antigen (see, for example, U.S. Patent No. 5,690,938 ). &Lt; / RTI &gt; Systemic administration of the targeted immunogen can also produce mucosal immunization (U.S. Patent No. 5,518,725). Various strategies have also been described for specific targeting of chimeric virus (see, e.g., U.S. Patent No. 5,714,374), adenovirus, vaccinia virus, or nucleic acid (see, for example, PCT Application No. WO 97/05267 Lt; RTI ID = 0.0 &gt; mucosal &lt; / RTI &gt;

For oral administration, the formulations of the present invention include, for example, binders (e.g., pregelatinised corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); Fillers (e. G., Lactose, microcrystalline cellulose or calcium hydrogen phosphate); Lubricants (e. G., Magnesium stearate, talc or silica); Disintegrants (e. G., Potato starch or sodium starch glycolate); Or in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as wetting agents (e. G., Sodium lauryl sulfate). The tablets may be coated by methods well known in the art. The compositions of the present invention may also be incorporated into microspheres or microcapsules assembled from, for example, poly-glycolic acid / lactic acid (PGLA) (see U.S. Patent Nos. 5,814,344; 5,100,669 and 4,849,222; PCT Publication Nos. WO 95/11010 and WO 93/07861). Liquid preparations for oral administration may take the form of, for example, solvents, syrups, emulsions or suspensions, or they may be presented as anhydrous products for reconstitution with water or other suitable vehicle before use. The liquid preparations may contain suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); Emulsifiers (e. G., Lecithin or acacia); Non-aqueous vehicles (e. G., Almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); And preservatives (e. G., Methyl or propyl-p-hydroxybenzoates or sorbic acid). &Lt; / RTI &gt; The formulations may also suitably contain buffer salts, flavoring agents, coloring agents and sweetening agents. Formulations for oral administration may be suitably formulated to provide controlled release of the active compound.

For administration by inhalation, the therapeutic agent according to the present invention may be administered in combination with a suitable propellant, e. G. Dichlorodifluoro-methane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, From a pack or sprayer to an aerosol spray delivery form. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for delivering the metered amount. For example, capsules and cartridges of gelatin containing a compound and a powder mixture of a suitable powder base such as lactose or starch may be formulated for use in an inhaler or an air injector.

The compositions of the present invention may also be formulated in rectal compositions such as suppositories, or rectal bowel preparations containing conventional suppository bases such as, for example, cocoa butter or other glycerides.

In addition to the formulations described previously, the compositions may also be formulated as a depot preparation. The long lasting formulations can be administered by implantation (e. G., Subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound may be formulated with a suitable polymeric or hydrophobic material (e.g., an acceptable oil in emulsion) or an ion exchange resin, or as an insoluble derivative, for example, as a poorly soluble salt have.

As described herein, the glycoprotein lipase (GSL) adjuvant with an antigen and / or a-glucose ([alpha] -Glc) can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, buffered saline, dextrose, glycerol, ethanol, sterile isotonic aqueous buffer, and the like and combinations thereof. In addition, if desired, the formulation may also contain an immunostimulant (glycoprotein having a-glucose ([alpha] -Glc) that enhances the efficacy of a wetting or emulsifying agent, a pH buffering agent and / GSL)). &Lt; / RTI &gt; Non-limiting examples of additional immunostimulatory agents that may enhance the effectiveness of the compositions of the present invention include immunostimulatory, immunostimulatory, or inflammation-promoting cytokines, lymphokines or chemokines, or nucleic acids encoding them (specific examples include interleukins Macrophage colony stimulating factor (CSF) and other colony stimulating factors, macrophage inflammatory factors, IL-1, IL-2, IL-3, IL-4, IL- Including Flt3 ligand, see further examples of immunostimulatory cytokines in the section titled "Definitions &quot;). These additional immunostimulatory molecules may be delivered systemically or locally, either as a protein or by expression of a vector encoding the expression of the molecule. The techniques described above for the delivery of glycoprotein high-quality (GSL) adjuvants with antigens and alpha -glucose ([alpha] -Glc) can also be used for the delivery of additional immunostimulatory molecules.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the immunogenic formulations of the invention. In a related aspect, the present invention provides a kit for the manufacture of a pharmaceutical or vaccine composition comprising one or more antigens and a glycoprotein-rich adipose (GSL) -containing adjuvant with? -Glucose (? -Glc) The kit comprises an antigen in a first container and a supplement for the second container, and optionally instructions for mixing and / or administering the composition. Each container of the kit may also optionally comprise one or more physiologically acceptable carriers and / or excipients and / or adjuvants. There may be an announcement in the form prescribed by a governmental agency regulating the manufacture, use or sale of a pharmaceutical or biological product in connection with said container (s), said notice being made by the manufacturer, Reflect the approval.

If desired, the composition may contain one or more unit dosage forms containing the active ingredient (i. E., A glycoprotein lipid (GSL) -containing adjuvant with an antigen and / or a-glucose Lt; / RTI &gt; pack or dispenser device. The pack may comprise, for example, a metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for treatment of a specified condition.

Effective capacity and safety assessment

In accordance with the methods of the present invention, the pharmaceutical and vaccine compositions described herein are preferably administered to a patient in an immunologically effective amount with minimal toxicity. An "immunologically effective amount" or "therapeutically effective amount" of the disclosed formulations is sufficient to produce an effective immune response in the treated subject, and therefore, (GSL) adjuvant with sufficient antigen and / or alpha -glucose ([alpha] -Glc).

After a well-established method in the art (see, for example, reports on evaluation of several vaccine formulations containing a novel collaborative effort between the Center for Biological Evaluation and Food and Drug Administration and the National Institute of Allergy and Infectious Diseases Effective dosing and toxicity of the compounds and compositions of the present invention may be achieved by first administering an antigen and a-glucose (&lt; RTI ID = 0.0 &gt; a-glucose &lt; / RTI &gt; (GSL) -containing adjuvant with both the GSL-containing adjuvant and the GSL-containing adjuvant, which has been found to be immunogenic and which can be reproducibly immunized by the same route proposed for human clinical trials ) In the preclinical study. Specifically, for the pharmaceutical compositions or vaccines used in the method of the invention, the therapeutically effective amount is the first, IC ₅₀ circulating plasma concentration range that includes a (i.e., the concentration of the test compound to achieve half-maximal inhibition of symptoms) &Lt; / RTI &gt; from an animal model to achieve the desired effect. A dose-response curve derived from the animal system is then used to determine test doses for initial clinical studies in humans. In determining safety for each composition, the dose and frequency of immunization should meet or exceed those expected for use in clinical trials.

As disclosed herein, the dose of glycocalgia (GSL), antigen (s) and other ingredients with alpha -glucose (alpha -Glc) in the compositions of the present invention can be adjusted continuously or intermittently Of the test animal and the individual conditions. The particular amount will vary naturally depending on the route of administration, the condition of the patient or the subject animal, such as age, weight, sex, irritability, feed, duration of administration, drug used in the formulation, severity of the disease. Under certain conditions, the appropriate dose and time of administration may be determined by testing based on the indications described above and should be determined according to the circumstances of each patient according to the judgment of the practitioner and standard clinical techniques. In this regard, the capacity of the antigen generally ranges from 0.1 μg to 100 mg per kg of body weight, and the glycoprotein quality (GSL) with α-glucose (α-Glc) required to enhance the immune response to the antigen, The dose of adjuvant is generally in the range of 10 to 100 [mu] g per kg of body weight.

The toxic and therapeutic efficacy of the glycocalypse rich (GSL) -containing immunogenic compositions with [alpha] -Glc of the present invention can be determined by standard pharmaceutical procedures in experimental animals, for example, LD ₅₀ 50% lethal dose of the population) and the ED ₅₀ (the therapeutically effective dose at 50% of the population). The dose ratio between toxic and therapeutic effects is a therapeutic index and this can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compositions exhibiting a large therapeutic index are preferred. Although therapeutic agents exhibiting toxic side effects can be used (e.g., when treating severe forms of cancer or life-threatening infection), the immunogenic compositions can be used to minimize potential damage to other tissues and organs, Care should be taken to design a delivery system that targets a particular site (for example, a lymphatic tissue that mediates an immune response, a tumor of an infectious agent, or an organ-assisted replication). As disclosed herein (see also background section and examples), the glycoprotein high-grade (GSL) adjuvant with? -Glucose (? -Glc) of the present invention has relatively low doses 10-100 [mu] g of sugar supplements), although highly immunostimulatory, possesses low toxicity and does not produce significant side effects.

As specified above, the data obtained from animal studies can be used to indicate a dosage range for use in humans. Glycidyl course having α- glucose (α-Glc) a therapeutically effective amount of the human ping lipid (GSL) - containing composition is in the circulating concentration range that includes that preferably, have a substantially toxic ED _50. The dose may vary within the above ranges depending on the dosage form used and the route of administration used. Ideally, a single capacity should be used.

Definitions indicated in the chemical structure. Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified according to the literature ([Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed., Inside cover], and certain functional groups are generally defined as described herein. In addition, the general principles of organic chemistry as well as specific functionality moieties and reactivity can be found in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 ^rd Edition, Cambridge University Press, Cambridge, 1987.

The compounds described herein may have one or more asymmetric centers and thus may exist in various isomeric forms, e. G., Enantiomers and / or diastereoisomers. For example, the compounds described herein may exist in the form of individual enantiomers, diastereomers or geometric isomers or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be separated from the mixture by methods known to those skilled in the art, including, for example, chiral high pressure liquid chromatography (HPLC) and formation and crystallization of chiral salts; Preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33: 2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, Ed., Univ. Of Notre Dame Press, Notre Dame, I N 1972). The disclosure of the present invention further encompasses the compounds described herein as individual isomers substantially free of other isomers and alternatively as a mixture of various isomers.

When listing a range of values, it is intended that each value and sub-range is included in the range. For example, "C _{1 -6"} is _{C 1, C 2, C 3} , C 4, C 5, C 6, C 1 -6, C 1 -5, C 1 -4, C 1 -3, C _{1 -2, C 2 -6, C} 2 -5, C 2 -4, C 2 -3, C 3 -6, C 3 -5, C 3 -4, C 4 -6, C 4 -5, and It is intended to include the C _{5 -6.}

"Alkyl" refers to a radical of a straight or branched saturated hydrocarbon group having from 1 to 20 carbon atoms ("C _{1 -20} alkyl"). In certain embodiments, the alkyl group has 1 to 10 carbon atoms ( "C _{1 -10} alkyl"). In certain embodiments, the alkyl group has from 1 to 9 carbon atoms ( "C _{1 -9} alkyl"). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C _1-8 alkyl"). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C _1-7 alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C _1-6 alkyl"). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C _1-5 alkyl"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C _1-4 alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C _1-3 alkyl"). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C _1-2 alkyl"). In some embodiments, the alkyl group has one carbon atom ("C ₁ alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C _{2 -6} alkyl"). Examples of C _1-6 alkyl groups include methyl (C _1), ethyl (C _2), n- propyl (C _3), iso-propyl (C _3), n- butyl (C _4), 3-tert-butyl ( C _4), 2-tert-butyl (C _4), iso-butyl (C _4), n- pentyl (C _5), 3- fentanyl (C _5), amyl (C _5), neopentyl (C _5), 3-methyl-2-butanyl (C ₅ ), tertiary amyl (C ₅ ), and n-hexyl (C ₆ ). Further examples of alkyl groups include n-heptyl (C ₇ ), n-octyl (C ₈ ), and the like. Unless otherwise specified, each instance of an alkyl group is optionally independently substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). In certain embodiments, the alkyl group is unsubstituted C _{1 -10} alkyl (e.g., -CH ₃ ). In certain embodiments, the alkyl group is a substituted C _{1 -10} alkyl.

"Alkenyl" refers to radicals of straight or branched chain hydrocarbon groups having from 2 to 20 carbon atoms, at least one carbon-carbon double bond and no triple bond ("C _2-20 alkenyl"). In some embodiments, the alkenyl group has 2 to 10 carbon atoms ("C _2-10 alkenyl"). In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C _2-9 alkenyl"). In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C _2-8 alkenyl"). In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C _2-7 alkenyl"). In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C _2-6 alkenyl"). In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C _2-5 alkenyl"). In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("C _2-4 alkenyl"). In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("C _2-3 alkenyl"). In certain embodiments, the alkenyl group has 2 carbon atoms ( "C ₂ alkenyl"). One or more carbon-carbon double bonds may be internal (as in 2-butenyl) or at the end (as in 1-butenyl). Examples of C _{2 -4} alkenyl groups include ethenyl (C ₂ ), 1-propenyl (C ₃ ), 2-propenyl (C ₃ ), 1-butenyl (C ₄ ) ₄ ), butadienyl (C ₄ ), and the like. Examples of C _{2 -6} alkenyl groups include pentenyl (C ₅ ), pentadienyl (C ₅ ), hexenyl (C ₆ ) and the like as well as the above-mentioned C _{2 -4} alkenyl groups. Further examples of alkenyl include heptenyl (C ₇ ), octenyl (C ₈ ), octatrienyl (C ₈ ), and the like. Unless otherwise specified, each occurrence of the alkenyl group is optionally independently substituted, i. E., Unsubstituted or substituted ("unsubstituted alkenyl") with one or more substituents ("substituted alkenyl"). In certain embodiments, the alkenyl group is a _2-10 alkenyl al unsubstituted C. In certain embodiments, the alkenyl group is an alkenyl-substituted _2-10 C.

"Alkynyl" refers to a radical of a straight or branched chain hydrocarbon group having from 2 to 20 carbon atoms, at least one carbon-carbon triple bond, and optionally at least one double bond ("C _2-20 alkynyl & . In some embodiments, the alkynyl group has 2 to 10 carbon atoms ("C _2-10 alkynyl"). In some embodiments, the alkynyl group has 2 to 9 carbon atoms ("C _2-9 alkynyl"). In some embodiments, the alkynyl group has 2 to 8 carbon atoms ("C _2-8 alkynyl"). In some embodiments, the alkynyl group has 2 to 7 carbon atoms ("C _2-7 alkynyl"). In some embodiments, the alkynyl group has 2 to 6 carbon atoms ("C _2-6 alkynyl"). In some embodiments, the alkynyl group has 2 to 5 carbon atoms ("C _2-5 alkynyl"). In some embodiments, the alkynyl group has 2 to 4 carbon atoms ("C _2-4 alkynyl"). In some embodiments, the alkynyl group has 2 to 3 carbon atoms ("C _2-3 alkynyl"). In some embodiments, the alkynyl group has two carbon atoms ("C ₂ alkynyl"). One or more carbon-carbon triple bonds may be internal (as in 2-butynyl) or at the end (such as in 1-butynyl). Examples of C _{2 -4} alkynyl group are, without limitation, ethynyl (C _2), 1- propynyl (C _3), 2- propynyl (C _3), 1- butynyl (C _4), 2- Butynyl (C ₄ ), and the like. Examples of C _{2 -6} alkenyl groups include pentynyl (C ₅ ), hexynyl (C ₆ ) and the like, as well as the C _{2 -4} alkynyl groups mentioned above. Further examples of alkynyl include heptynyl (C ₇ ), octynyl (C ₈ ), and the like. Unless otherwise specified, each occurrence of the alkynyl group is optionally independently substituted, i.e., unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents ("substituted alkynyl"). In certain embodiments, the alkynyl group is an unsubstituted C _{2 -10} alkynyl. In certain embodiments, the alkynyl group is a substituted C _{2 -10} alkynyl.

"Carbocyclyl", "carbocyclic" or non-refers to a radical of an aromatic hydrocarbon group ( "C _{3 -10-non-aromatic} having from 3 to 10 ring carbon atoms and zero heteroatoms in the ring system Carbocyclyl "). In some embodiments, carbocyclyl groups can have from 3 to 8 ring carbon atoms ( "C _{3 -8} carbocyclyl"). In some embodiments, the carbocyclyl group has 3 to 6 ring carbon atoms ("C _{3 -6} carbocyclyl"). In some embodiments, the carbocyclyl group has 3 to 6 ring carbon atoms ("C _{3 -6} carbocyclyl"). In some embodiments, the carbocyclyl group has 5 to 10 ring carbon atoms ("C _5-10 carbocyclyl"). Examples of C _{3 -6} carbocyclyl groups are, without limitation, cyclopropyl (C _3), cyclopropyl-propenyl (C _3), cyclobutyl (C _4), cyclo-butenyl (C _4), cyclopentyl (C ₅ ), Cyclopentenyl (C ₅ ), cyclohexyl (C ₆ ), cyclohexenyl (C ₆ ), cyclohexadienyl (C ₆ ), and the like. Examples of C _{3 -8} carbocyclyl groups include, but are not limited to, the above-mentioned C _3-6 carbocyclyl groups as well as cycloheptyl (C ₇ ), cycloheptenyl (C ₇ ), cycloheptadienyl (C ₇ ), tart Rie cycloheptane carbonyl (C _7), cyclooctyl (C _8), cyclo octenyl (C _8), hydroxy [2.2.1] heptanyl (C _7), hydroxy [2.2.2] octa-carbonyl (C ₈ ), and the like. C _{3 -10} carbonyl Examples of heterocyclyl groups are, without limitation, the above-mentioned C _{3 -8} carbocyclyl groups as well as cyclo-nonyl (C _9), none cycloalkyl carbonyl (C _9), cycloalkyl-decyl (C _10), (C ₁₀ ), octahydro-1H-indenyl (C ₉ ), decahydronaphthalenyl (C ₁₀ ), spiro [4.5] decanyl (C ₁₀ ) and the like. As described in the foregoing examples, in certain embodiments, the carbocyclyl group is a monocyclic ("monocyclic carbocyclyl") or fused, bridged or spirocyclic ring system, such as a bicyclic ring system Bicyclic carbocyclyl ") and may be saturated or partially unsaturated. "Carbocyclyl" also includes a ring system wherein the carbocyclic ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on a carbocyclic ring , In which case the number of carbons continues to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each occurrence of the carbocyclyl group is optionally independently substituted, i.e., unsubstituted (or "unsubstituted carbocyclyl") is substituted with one or more substituents ("substituted carbocyclyl "). In certain embodiments, the carbocyclyl group is a non-substituted C _{3 -10} carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C _{3 -10} carbocyclyl.

In some embodiments, "carbocyclyl" is a monocyclic, saturated carbocyclyl group having 3 to 10 ring carbon atoms ("C _{3 -10} cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ( "C _{3 -8} cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 6 ring carbon atoms ("C _{3 -6} cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 6 ring carbon atoms ("C _5-6 cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 10 ring carbon atoms ( "C _{5 -10} cycloalkyl"). Examples of C _{5 -6} cycloalkyl groups include cyclopentyl (C _5), and cyclohexyl (C _5). Examples of C _{3 -6} cycloalkyl group as well as C _{5 -6} cycloalkyl groups referred to above include cyclopropyl (C ₃₎ and cyclobutyl (C _4). Examples of C _{3 -8} cycloalkyl group, as well as C _{3 -6} cycloalkyl groups referred to above include cycloheptyl (C ₇₎ and cyclooctyl (C _8). Unless otherwise specified, each occurrence of the cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, the cycloalkyl group is a C _3-10 cycloalkyl which is unsubstituted. In certain embodiments, the cycloalkyl group is a substituted C _{3 -10} cycloalkyl.

"Heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 10-membered non-aromatic ring system having a ring carbon atom and from 1 to 4 ring heteroatoms, wherein each heteroatom is independently Nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3-10 membered heterocyclyl"). In certain embodiments, the heteroatoms are independently selected from nitrogen, sulfur, and oxygen. In heterocyclyl groups containing at least one nitrogen atom, the attachment point may be a carbon or nitrogen atom, so long as the valence permits. The heterocyclyl group may be monocyclic ("monocyclic heterocyclyl") or may be a fused, bridged or spirocyclic system, such as a bicyclic system ("bicyclic heterocyclyl"), And can be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may contain one or more heteroatoms in one ring or both rings. "Heterocyclyl" also includes a ring system wherein the heterocyclic ring as defined above is fused with one or more carbocyclyl groups, wherein the attachment point is a carbocyclyl or a heterocyclic ring, or Wherein the heterocyclic ring as defined above is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on a heterocyclic ring and in this case the ring member The number continues to designate the number of ring members in the heterocyclic ring system. Unless otherwise specified, each occurrence of the heterocyclyl is optionally substituted independently, i.e., unsubstituted ("unsubstituted heterocyclyl") is substituted with one or more substituents ("substituted heterocyclyl" ). In certain embodiments, the heterocyclyl group is an unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

In some embodiments, the heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron , Phosphorus, and silicon ("5- to 10-membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-8 membered non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 8-membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heterocyclyl"). In some embodiments, the 5- to 6-membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary three-membered heterocyclyl groups containing one heteroatom include, without limitation, aziridinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, And pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxysulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazininyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocannyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups (also referred to herein as 5,6-bicyclic heterocyclic rings) fused to C ₆ aryl rings include, without limitation, indolinyl, isoindolinyl, dihydro Benzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclic rings) fused to an aryl ring include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl And the like.

Refers to a monocyclic or polycyclic (e. G., Bicyclic or tricyclic) 4n + 2 aromatic ring system having 6-14 ring carbon atoms and 0 heteroatoms in the aromatic ring system for example, it refers to a radical of the 6, 10, or 14 having a π electron) shared in a cyclic array ( "C _{6 -14} aryl"). In some embodiments, the aryl group has 6 ring carbon atoms ("C ₆ aryl &quot;, e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C ₁₀ aryl"; eg, naphthyl, for example, 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (" _C14 aryl &quot;; e.g., anthracyl). "Aryl" also includes a ring system wherein an aryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, wherein the radical or attachment point is on an aryl ring, , The number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each occurrence of the aryl group is optionally independently substituted, i. E., Unsubstituted or substituted ("unsubstituted aryl") with one or more substituents ("substituted aryl"). In certain embodiments, the aryl group is unsubstituted C _6-14 aryl. In a particular embodiment, the aryl group is a substituted C _{6 -14} aryl group.

"Arylalkyl" is a subset of alkyl and aryl as defined herein, and refers to an optionally substituted alkyl group substituted by an optionally substituted aryl group. In certain embodiments, the aralkyl is an optionally substituted benzyl. In certain embodiments, the aralkyl is benzyl. In certain embodiments, the aralkyl is optionally substituted phenethyl. In certain embodiments, the aralkyl is phenethyl.

"Heteroaryl" means a 5-10 membered monocyclic or bicyclic 4n + 2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system (e. G., Cyclic Quot; heteroaryl "), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heteroaryl"). In a heteroaryl group containing at least one nitrogen atom, the point of attachment may be a carbon or nitrogen atom, so long as the valence permits. A heteroaryl bicyclic ring system may contain one or more heteroatoms in one ring or both rings. "Heteroaryl" includes ring systems wherein a heteroaryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on a heteroaryl ring, , The number of ring members continues to designate the number of ring members in the heterocyclic ring system. "Heteroaryl" also includes a ring system wherein a heteroaryl ring as defined above is fused with one or more aryl groups, wherein the point of attachment is on an aryl or heteroaryl ring, The number of members designates the number of ring members in the fused (aryl / heteroaryl) ring system. In the bicyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment may be in the ring, i.e., a ring containing a heteroatom (For example, 2-indolyl) or a ring containing no heteroatom (for example, 5-indolyl).

In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in an aromatic ring system, wherein each heteroatom is independently nitrogen, oxygen, and Sulfur "(" 5- to 10-membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in an aromatic ring system, wherein each heteroatom is independently nitrogen, oxygen, and Sulfur " ("5- to 8-membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in an aromatic ring system, wherein each heteroatom is independently nitrogen, oxygen, and Sulfur "(" 5-6 membered heteroaryl"). In some embodiments, the 5- to 6-membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has one ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each occurrence of the heteroaryl group is optionally independently substituted, i. E., Unsubstituted or substituted ("unsubstituted heteroaryl") with one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Examples of 7-membered heteroaryl groups containing one heteroatom include, without limitation, azetpinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benz Imidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and furunyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl .

"Heteroaralkyl" is a subset of alkyl and heteroaryl as defined herein, and refers to an optionally substituted alkyl group substituted by an optionally substituted heteroaryl group.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups as defined herein, wherein the divalent bridging group is a suffix-ene such as alkylene, alkenylene, Alkynylene, carbocyclylene, heterocyclylene, arylene, and heteroarylene.

The term "optionally substituted " as used herein refers to a substituted or unsubstituted moiety.

The alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups as defined herein are optionally substituted (e.g., "substituted" or " Quot; substituted "or" unsubstituted "heterocyclyl, " substituted" or " Cyclic, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl group). Generally, the term "substituted" means that at least one hydrogen (e.g., carbon or nitrogen atom) present on a group is replaced by an acceptable substituent, for example, a substituent Means that the compound is replaced by a substituent that results in a stable compound, for example, a compound that does not spontaneously undergo rearrangement, cyclization, elimination, or conversion by other reactions. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position is substituted in any given structure, the substituents are the same or different at each position. The term "substituted" is contemplated to include substitution of an organic compound with all permissible substituents, and any of the substituents described herein lead to the formation of a stable compound. The present invention contemplates any such combination to arrive at a stable compound. For purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents as described herein, resulting in the formation of a stable moiety by meeting the valency of the heteroatom.

Exemplary carbon atom substituents are _{halogen, -CN, -NO 2, -N 3} , -SO 2 H, -SO 3 H, -OH, -OR aa, -ON (R bb) 2, -N (R bb) ^{_{2, -N (R bb) 3}} + X -, -N (OR cc) R bb, -SH, -SR aa, -SSR cc, -C (= 0) R aa, -C0 2 H, -CHO, _{^{-C (OR cc) 2, -C0}} 2 R aa, -OC (= 0) R aa, -OC0 2 R aa, -C (= 0) N (R bb) 2, -OC (= 0) N ( ^{_{R bb) 2, -NR bb C}} (= 0) R aa, -NR bb C0 2 R aa, -NR bb C (= 0) N (R bb) 2, -C (= NR bb) R aa, - ^{C (= NR bb) OR aa} , -OC (= NR bb) R aa, -OC (= NR bb) OR aa, -C (= NR bb) N (R bb) 2, -OC (= NR bb) _{^{N (R bb) 2, -NR}} bb C (= NR bb) N (R bb) 2, -C (= 0) NR bb S0 2 R aa, -NR bb S0 2 R aa, -S0 2 N (R ^{_{bb) 2, -S0 2 R aa}} , -S0 2 OR aa, -OSO 2 R aa, -S (= 0) R aa, -OS (= 0) R aa, -Si (R aa) 3, -Osi _{^{(R aa) 3 -C (=}} S) N (R bb) 2, -C (= 0) SR aa, -C (= S) SR aa, -SC (= S) SR aa, -SC (= 0 ^{) SR aa, -OC (= 0} ) SR aa, -SC (= 0) OR aa, -SC (= 0) R aa, -P (= 0) 2 R aa, -OP (= 0) 2 R aa , -P (= 0) (R aa) 2, -OP (= 0) (R aa) 2, -OP (= 0) (OR cc) 2, -P (= 0) 2 N (R bb) 2 _{, -OP (= 0) 2 N} (R bb) 2, -P (= 0) (NR bb) 2, -OP (= 0) (NR bb) 2, -NR bb P (= 0) ^{_{(OR cc) 2, -NR bb}} P (= 0) (NR bb) 2, -P (R cc) 2, -P (R cc) 3, -OP (R cc) 2, -OP (R cc) ^{_{3, -B (R aa) 2}} , -B (OR cc) 2, -BR aa (OR cc), C 1 -10 alkyl, C _{1 -10} alkyl, perhaloalkyl, C _{2 -10} alkenyl to, C _{2 - 10} alkynyl, C _{3 -10} carbocyclyl, heterocyclyl of 3-14 won reel, including heteroaryl of C _{6 -14} aryl, and 5-14 won but not limited to, wherein each of the alkyl, al Alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; Or two hydrogens on the carbon atom is a group = 0, = S, = NN (R bb) 2, = NNR bb C (= 0) R aa, = NNR bb C (= 0) OR aa, = NNR bb S ( = O) ₂ R ^aa , = NR ^bb , or = NOR ^cc ; When each of R ^aa is independently C _{1 -10} alkyl, perhalo C _{1 -10} alkyl, C _{2 -10} alkenyl to, C _{2 -10} alkynyl, C _{3 -10} carbonyl of heterocyclyl, 3-14 won heterocyclyl, C _{6 -14} aryl, and 5-14 membered heteroaryl, or of, or two of the R groups are connected to ^aa form a heteroaryl ring of 3-14 membered heterocyclyl or 5-14 membered of Wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; When each of R ^bb are independently ^{hydrogen, -OH, -OR aa, -N (} R cc) 2, -CN, -C (= 0) R aa, -C (= 0) N (R cc) 2 ^{_{, -C0 2 R aa, -S0 2}} R aa, -C (= NR cc) OR aa, -C (= NR cc) N (R cc) 2, -S0 2 N (R cc) 2, -S0 2 ^{_{R cc, -S0 2 OR cc,}} -SOR aa, -C (= S) N (R cc) 2, -C (= 0) SR cc, -C (= S) SR cc, -P (= 0) ^{_{2 R aa, -P (= 0}} ) (R aa) 2, -P (= 0) 2 N (R cc) 2, -P (= 0) (NR cc) 2, C 1 -10 alkyl, C _{1 to-10} perhalo alkyl, C _{2 -10} alkenyl, C _{2 -10} alkynyl, C _{3 -10} carbocycle heteroaryl of the reel, 3-14 membered heterocyclyl, C _{6 -14} aryl, and 5-14 membered heteroaryl of Or two R ^bb groups are connected to form a 3-14 membered heterocyclyl or a 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl , Heterocyclyl, aryl, and heteroaryl are independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; Each occurrence of R ^cc is independently selected from the group consisting of hydrogen, C _{1 -10} alkyl, C _{1 -10} perhaloalkyl, C _{2 -10} alkenyl, C _{2 -10} alkynyl, C _{3 -10} carbocyclyl, heterocyclyl of the circle, C _{6 -14} aryl, and 5-14 membered heteroaryl, or in, or two R ^cc group of 5-14 membered heterocyclic and the 3-14 membered heteroaryl is connected to ring Wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups Being; When each of R ^dd is independently _{halogen, -CN, -N0 2, -N 3} , -S0 2 H, -S0 3 H, -OH, -OR ee, -ON (R ff) 2, -N ( _{^{R ff) 2, -N (R}} ff) 3 + X -, -N (OR ee) R ff, -SH, -SR ee, -SSR ee, -C (= 0) R ee, -C0 2 H, ^{_{-C0 2 R ee, -OC (=}} 0) R ee, -OC0 2 R ee, -C (= 0) N (R ff) 2, -OC (= 0) N (R ff) 2, -NR ff ^{C (= 0) R ee,} -NR ff C0 2 R ee, -NR ff C (= 0) N (R ff) 2, -C (= NR ff) OR ee, -OC (= NR ff) R ee ^{, -OC (= NR ff) OR} ee, -C (= NR ff) N (R ff) 2, -OC (= NR ff) N (R ff) 2, -NR ff C (= NR ff) N ( ^{_{R ff) 2, -NR ff S0}} 2 R ee, -S0 2 N (R ff) 2, -S0 2 R ee, -S0 2 OR ee, -OS0 2 R ee, -S (= 0) R ee, _{^{-Si (R ee) 3, -Osi}} (R ee) 3, -C (= S) N (R ff) 2, -C (= 0) SR ee, -C (= S) SR ee, -SC ( ^{= S) SR ee, -P (} = 0) 2 R ee, -P (= 0) (R ee) 2, -OP (= 0) (R ee) 2, -OP (= 0) (OR ee) _2, C _{1 -6} alkyl, C _{1 -6} alkyl, perhaloalkyl, C _2-6 alkenyl, C _{2 -6-alkynyl,} C _{3 -10} carbocyclyl, heterocyclyl of 3-10 won reel, C _{6 - 10} aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, Alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently substituted with 0, 1, 2, 3, 4, or 5 R ^gg groups, or two R ^dd substituents are joined to form = 0 Or = S; When each of R ^ee is independently an alkyl as C _{1 -6} alkyl, C _{1 -6} perhaloalkyl, C _{2 -6} alkenyl, C _{2 -6-alkynyl,} C _{3 -10} carbocyclyl, C _{6 -10} aryl , 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R ^gg groups; When each of R ^ff are independently hydrogen, C _{1 -6} alkyl, alkyl, C _{2 -6} alkenyl with C _1-6 perhaloalkyl, C _{2 -6-alkynyl,} C _{3 -10} carbocyclyl, 3-10 heterocyclyl of the circle, C _{6 -10} aryl, and a 5-10 membered, or heteroaryl, or two R ^ff group is a heteroaryl ring of 5-14 membered heterocyclic 3-13 membered connected Wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^gg groups ; When each of R ^gg is independently _{halogen, -CN, -N0 2, -N 3} , -S0 2 H, -SO 3 H, -OH, -OC 1 -6 alkyl, -ON (C _1-6 alkyl ) _2, -N (C _{1 -6} alkyl) _2, -N (C _{1 -6 ^{alkyl) 3 + X -, -NH (}} C 1 -6 ^{_{alkyl) 2 + X -, -NH 2}} (C 1 -6 ^{_{alkyl) + X -, -NH 3 +}} X -, -N (OC 1 -6 alkyl) (C _{1 -6} alkyl), -N (OH) (C 1 -6 alkyl), -NH (OH), - SH, -SC _{1 -6} alkyl, -SS (C _{1 -6} alkyl), -C (= 0) ( C 1 -6 _{alkyl), -C0 2 H, -C0 2} (C 1 -6 alkyl), - _{OC (= 0) (C 1} -6 alkyl), -OC0 ₂ (C _{1 -6} alkyl), -C (= 0) NH 2, -C (= 0) N (C 1-6 alkyl) _2, - OC (= 0) NH (C 1 -6 alkyl), -NHC (= 0) ( C 1-6 alkyl), -N (C _{1 -6} alkyl) C (= 0) (C 1 -6 alkyl), -NHC0 ₂ (C _{1 -6} alkyl), -NHC (= 0) N (C 1-6 _{alkyl) 2, -NHC (= 0)} NH (C 1 -6 alkyl), -NHC (= O) NH 2 , -C (= NH) O ( C 1-6 alkyl), -OC (= NH) ( C 1-6 alkyl), -OC (= NH) OC 1 -6 alkyl, -C (= NH) N ( C _{1-6 alkyl) 2, -C (= NH)} NH (C 1-6 alkyl), -C (= NH) NH 2, -OC (= NH) N (C 1 -6 alkyl) _2, -OC (NH) NH (C _{1 -6 alkyl), -OC (NH) NH 2} , -NHC (NH) N (C 1-6 _{alkyl) 2, -NHC (= NH)} NH 2, -NHSO 2 (C 1 _{-6 alkyl), -SO 2 N (C 1} -6 alkyl) _2, - SO ₂ NH (C _{1-6 alkyl), -SO 2 NH 2 -,} SO 2 C 1 -6 alkyl, -SO ₂ OC _{1 -6} alkyl, -OSO ₂ C _{1 -6} alkyl, -SOC _{1 -6} alkyl , -Si (C _{1 -6} alkyl) _3, -Osi (C _{1 -6 alkyl) 3 -C (= S) N} (C 1 -6 _{alkyl) 2, C (= S)} NH (C 1-6 alkyl ), C (= S) NH 2, -C (= 0) S (C 1-6 alkyl), -C (= S) SC 1-6 alkyl, -SC (= S) SC _1-6 alkyl, - _{P (= 0) 2 (C} 1-6 alkyl), -P (= 0) ( C 1 -6 _{alkyl) 2, -0P (= 0)} (C 1 -6 alkyl) _2, -OP (= 0) (OC _{1 -6} alkyl) _2, C _{1 -6} alkyl, C _{1 -6} alkyl, perhaloalkyl, C _{2 -6} alkenyl, C _{2 -6-alkynyl,} C _{3 -10} carbocyclyl, C _{6 -10} aryl , 3-10 membered heterocyclyl, 5-10 membered heteroaryl; Or two R &lt; ^g &gt; substituents may be joined to form = 0 or = S; Where X ^- is a counter ion.

Refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

The "acyl" as used herein is ^{-C (= 0) R aa,} -CHO, -C0 2 R aa, -C (= 0) N (R bb) 2, -C (= NR bb) R aa ^{, -C (= NR bb) OR} aa, -C (= NR bb) N (R bb) 2, -C (= 0) NR bb S0 2 R aa, -C (= S) N (R bb) 2 , -C (= O) SR ^aa , and -C (= S) SR ^aa , wherein R ^aa and R ^bb are as defined herein.

The nitrogen atom may be substituted or unsubstituted as far as the valence permits, and includes primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen substituent is ^{hydrogen, -OH, -OR aa, -N (} R cc) 2, -CN, -C (= 0) R aa, -C (= 0) N (R cc) 2, -C0 _{^{2 R aa, -S0 2 R aa}} , -C (= NR bb) R aa, -C (= NR cc) OR aa, -C (= NR cc) N (R cc) 2, -S0 2 N (R _{^{cc) 2, -S0 2 R cc}} , -S0 2 OR cc, -SOR aa, -C (= S) N (R cc) 2, -C (= 0) SR cc, -C (= S) SR cc _{, -P (= 0) 2 R} aa, -P (= 0) (R aa) 2, -P (= 0) 2 N (R cc) 2, -P (= 0) (NR cc) 2, C _1-10 alkyl, C _1-10 alkyl, perhaloalkyl, C _{2 -10} alkenyl, C _{2 -10} alkynyl, C _{3 -10} carbocyclyl, heterocyclyl of 3-14 won reel, C _{6 -14} aryl, And &lt; RTI ID = 0.0 &gt; 5-14 &lt; / RTI &gt; membered heteroaryl, or two R ^cc Groups are joined to form a 3-14 membered heterocyclyl or a 5-14 membered heteroaryl ring wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl Are independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups, wherein R ^aa , R ^bb , R ^cc , and R ^dd are as defined above.

In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups are ^{-OH, -OR aa, -N (R} cc) 2, -C (= 0) R aa, -C (= 0) N (R cc) 2, -C0 2 R aa, -S0 2 ^{R aa, -C (= NR cc} ) R aa, -C (= NR cc) OR aa, -C (= NR cc) N (R cc) 2, -S0 2 N (R cc) 2, -S0 2 _{^{R cc, -S0 2 OR cc,}} -SOR aa, -C (= S) N (R cc) 2, -C (= 0) SR cc, -C (= S) SR cc, C 1 -10 alkyl ( for example, aralkyl, heteroaralkyl), C _{2 -10} alkenyl, C _{2 -10} alkynyl, C _{3 -10} carbocyclyl, 3-14 membered heterocyclyl, C _{6 -14} aryl, and Wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently selected from 0, 1, , 2, 3, 4, or 5 R ^dd groups, wherein R ^aa , R ^bb , R ^cc, and R ^dd are as defined herein. Nitrogen protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 ^rd edition, John Wiley & Sons, 1999, .

For example, a nitrogen protecting group such as an amide group (e.g., -C (= O) R ^aa ) may be protected with a protecting group such as formamide, acetamide, chloroacetamide, trichloroacetamide, But are not limited to, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanine derivatives, benzamide, p- 3- (p-hydroxyphenyl) propanamide, 3- (o-nitrophenyl) propanamide, 2-methyl-2- (o-nitrophenoxy) propanamide, 2-methyl-2- (o-phenylazophenoxy) propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o- N-acetylmethionine derivatives, o-nitrobenzamide, and o- (benzoyloxymethyl) benzamide But not limited thereto.

A nitrogen protecting group such as a carbamate group (e. G. -C (= O) OR ^aa ) can be prepared by reacting methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9- -Sulfo) fluorenylmethyl carbamate, 9- (2,7-dibromo) fluoroenylmethyl carbamate, 2,7-di-t-butyl- [9- (10,10- (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc) , 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl) -1- methylethyl (Adpoc), 1,1- Dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2- trichloroethyl carbamate (TCBOC), 1- (2 ', 4'-di-tert-butylphenyl) -1-methylethylcarbamate (t-Bumeoc) - and 4'-pyridyl) ethyl carbamate (Pyoc) , 2- (N, N-dicyclohexylcarboxamido) ethyl carbamate, t-butyl carbamate (BOC), 1- adamantyl carbamate (Adoc), vinyl carbamate (Voc) (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamylcarbamate (Coc), 4-nitrocinnamylmercarbamate (Noc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate , Alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, -Dichlorobenzyl carbamate, 4-methylsulfinyl benzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonyl ethyl carbamate, 2 - (p-toluenesulfonyl) ethylcarbamate, [2- (1,3-dithianyl)] methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc) (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonium isopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m- (Dihydroxybaryl) benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2- (trifluoromethyl) -6-chromoylmethylcarbamate (Tcroc) , m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinylcarbamate, o- (N, N-dimethylcarboxamido) Benzyl carbamate, 1,1-dimethyl-3- (N, N-dimethylcarboxamido) propyl carbamate, 1,1-dimethylpropyl carbamate, di (2- pyridyl) methyl carbamate, 2- Isobutylcarbamate, isonicotinyl carbamate, p- (p'-methoxyphenyl azo) benzylcarbamate, 1-methylcyclohexylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, Butylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1- (3,5-dimethoxyphenyl) ethylcarbamate, (4-pyridyl) ethylcarbamate, phenylcarbamate, p- (phenylazo) benzyl, 3,5- But are not limited to, carbamates, 2,4,6-tri-t-butylphenyl carbamate, 4- (trimethylammonium) benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate. It is.

A nitrogen protecting group, for example, a sulfonamide group (e.g., -S (= O) 2R ^aa ) is reacted with p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, Methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6- (Mts), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzene (SMS), &lt; RTI ID = 0.0 &gt; 9-anthracene &lt; / RTI &gt; But are not limited to, sulfonamides, 4- (4 ', 8'-dimethoxynaphthylmethyl) benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide and phenacylsulfonamide.

Other nitrogen protecting groups include phenothiazinyl- (10) -acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanine derivatives, N-acetylmethionine derivatives , 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N- Dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan- , 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-hydroxyl, N- N-3-acetoxypropylamine, N- (1-isopropyl-4-nitro-2-oxo-3-pyrroline (4-methoxyphenyl) methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr) N - [(4-methoxyphenyl) diphe (Fcm), N-2-methyl-2-pyrrolidinylmethylamine (MMTr), N-9-phenylfluorenylamine (PhF) N, N'-diphenylmethyleneamine, N - [(2-pyridyl) mesitylene, N-diphenylmethylenediamine, N- (N ', N'-dimethylaminomethylene) amine, N, N'-isopropylidene diamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5- N-cyclohexylideneamine, N- (5,5-dimethyl-3-oxo-1-cyclohexenyl) amine , N-borane derivatives, N-diphenylboronic acid derivatives, N- [phenyl (pentaacylchromium- or tungsten) acyl] amine, N-copper chelate, N-zinc chelate, N-nitroamine, , Amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthio (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzene sulfenamide, o-nitrobenzene sulfenamide (Nps), 2,4-dinitrobenzene sulfenamide Amide, pentachlorobenzene sulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridine sulfenamide (Npys).

In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting group ^{-R aa, -N (R bb)} 2, -C (= 0) SR aa, -C (= 0) R aa, -C0 2 R aa, -C (= 0) N (R bb ^{_{) 2, -C (= NR bb}} ) R aa, -C (= NR bb) OR aa, -C (= NR bb) N (R bb) 2, -S (= 0) R aa, -S0 2 R _{^{aa, -Si (R aa) 3}} , -P (R cc) 2, -P (R cc) 3, -P (= 0) 2 R aa, -P (= 0) (R aa) 2, -P (= 0) (OR ^cc ) ₂ , -P (= O) ₂ N (R ^bb ) ₂ and -P (= 0) (NR ^bb ) ₂ , wherein R ^aa , R ^bb , and ^Rcc are as defined herein. Oxygen protecting groups are well known in the art and include those described in Poteking Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 ^rd edition, John Wiley & Sons, 1999, .

Exemplary oxygen protecting groups include but are not limited to methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl Butoxymethyl (POM), siloxymethyl (POM), 4-methoxyphenoxy, methyl (p-AOM), guaiacol methyl (GUM), t- , 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis (2- chloroethoxy) methyl, 2- (trimethylsilyl) ethoxymethyl (SEMOR), tetrahydropyran (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4 4-methoxypiperidin-4-yl (CTMP), 1, 4-dioxane-1, 2-dihydro- 2-yl, tetrahydrofuranyl, tetrahydrothiopyranyl, 2,3,3a, 4,5,6,7,7a 2-yl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 1-methyl-1-methoxy Methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl) Ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p- methoxybenzyl, 3,4-dimethoxybenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, And examples thereof include diphenylmethyl, p, p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p- methoxyphenyldiphenylmethyl, di (p- 4,4 ', 4 "-tris (4,5-dichloro-phthalimidophenyl) phenyl) methyl, Methyl, 4,4 ', 4 "-tris (levulinoyl Bis (4 ', 4 "-dimethoxyphenyl) methyl, 1,1-bis (trifluoromethyl) (9-phenyl-10-oxo) anthryl, 1,3-benzo [epsilon] (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethyl Butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), i-butyl methoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, Citrate, p-chlorophenoxy Acetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4- (ethylene dithio) pentanoate (levulonyl dithioacetal), pivaloate, Benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc) , Ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2- (trimethylsilyl) ethyl carbonate (TMSEC), 2- (phenylsulfonyl) ethyl carbonate (Psec) O) is at least one selected from the group consisting of ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC), p-nitrophenyl carbonate, benzyl carbonate, p- methoxybenzyl carbonate, , o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl 4-methylbutylate, 4-nitro-4-methylpentanoate, o- (dibromomethyl) methylenebis Benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy) ethyl, 4- (methylthiomethoxy) butyrate, 2- (methylthiomethoxymethyl) benzoate, 2,6- 4-methylphenoxyacetate, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl) phenoxyacetate, 2,4-bis (1,1-dimethylpropyl) phenoxyacetate, (Methoxyacyl) benzoate,? -Naphthoate, nitrate, alkyl N, N, N-diethylacetate, isobutyrate, N, N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfate, sulfate, methanesulfonate Rate), benzyl sulfonate, and tosylate (Ts).

In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups are ^{-R aa, -N (R bb)} 2, -C (= 0) SR aa, -C (= 0) R aa, -C0 2 R aa, -C (= 0) N (R bb ^{_{) 2, -C (= NR bb}} ) R aa, -C (= NR bb) OR aa, -C (= NR bb) N (R bb) 2, -S (= 0) R aa, -S0 2 R _{^{aa, -Si (R aa) 3}} , -P (R cc) 2, -P (R cc) 3, -P (= 0) 2 R aa, -P (= 0) (R aa) 2, -P (= 0) (OR ^cc ) ₂ , -P (= O) ₂ N (R ^bb ) ₂ and -P (= 0) (NR ^bb ) ₂ , wherein R ^{aa ,} R ^bb , and ^Rcc are as defined herein. The sulfur protecting groups are well known in the art and include those described in Protecintg Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 ^rd edition, John Wiley & Sons, 1999, .

As used herein, the term "leaving group " is its conventional meaning as set forth in the field of synthetic organic chemistry and refers to an atom or group that can be replaced by a nucleophile. Examples of suitable leaving groups include, but are not limited to, halogen (e.g., F, Cl, Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, But are not limited to, hydroxy (for example, acetoxy), arylcarbonyloxy, aryloxy, methoxy, N, O-dimethylhydroxylamino, In some cases, the leaving group may be selected from the group consisting of sulfonic acid esters, such as toluene sulfonate (tosylate, -OTs), methanesulfonate (mesylate, -OMs), p-bromobenzenesulfonyloxy OBs), or trifluoromethanesulfonate (triflate, -OTf). In some cases, the leaving group is a bromosylate, for example p-bromobenzenesulfonyloxy. In some cases, the leaving group is a nosylate, for example, 2-nitrobenzenesulfonyloxy. In some embodiments, the leaving group is a sulfonate-containing group. In some embodiments, the leaving group is a tosylate group. The leaving group may also be a phosphine oxide (e.g. formed during the Mitsunobu reaction) or an internal leaving group, e. G., Epoxide or cyclic sulfate. Other non-limiting examples of leaving groups are water, ammonia, alcohols, ether moieties, thioether moieties, zinc halides, magnesium moieties, diazonium salts, and copper moieties.

Exemplary [alpha] -GalCer analogs (GSL with [alpha] -Glc) are used as immunological adjuvants to accelerate, enhance, prolong and / or modify or augment the effect of the vaccine by stimulating the immune system of the vaccinated patient . In an exemplary run, analog C34 is used as an adjuvant. As used herein, the term "alum adjuvant" refers to an aluminum salt with an immunostimulatory activity that has immunosuppressive activity, such as aluminum phosphate and aluminum hydroxide. These exemplary agents can adsorb and precipitate protein antigens in solution; The resulting precipitate improves vaccine immunogenicity by promoting the release of antigen from the vaccine depot formed at the inoculation site. In addition, the adjuvants contemplated herein may also include suitable organic adjuvants and suitable virosomes. In certain embodiments, exemplary organic adjuvants may include oil based adjuvants such as squalene, MF59, QS-21 and AS03.

As used herein, the term " anti-tumor immunotherapeutic &lt; RTI ID = 0.0 &gt; activator &quot; refers &lt; / RTI &gt; to an antibody produced by the present vaccine that inhibits, reduces and / or eliminates the tumor, alone and /

The α-galactosyl group (αGal) on the acyl chain and the glycocopherol (GSL) containing the phenyl ring are more potent than the α-galactosylceramide (αGalCer), and both mouse and human invariant NKT It is known to stimulate differently. Their activity in mice and humans is related to the binding of the three-way interactions between the iNKT TCR and the CDld-GSL complex.

The disclosure of the present invention is based on the unexpected finding that GSL with glucose ([alpha] Glc) is stronger than having [alpha] Gal for humans in the induction of cytokines / chemokines and expansion / activation of immune cells, . The effects of F derivatives of GSL and aGlc with glucose ([alpha] Glc), and their immunostimulation in humans, are disclosed herein. Immune-stimulating effects associated with the intensity of the three-way interaction for each species are disclosed herein. Is an iNKT TCR rather than a CD 1d that directs a species-specific response as evidenced by the mCDld vs. hCDld swapping assay disclosed herein. Glycoprotein high-grade (GSL) with αGlc has stronger tertiary interactions in humans than GSL with αGal and induces greater Th1 -weight immunity. GSL with alpha Glc is less irritating than GSL with alpha Gal in mice. The species-specific response is due to the differential binding of species-terrestrial complexes and reflects the difference between mouse and human iNKT TCR as supported by the hCDld swapping test versus mCDld as described herein.

These novel discoveries show differences in species and provide a novel design of modified GSL on glucosyl groups and are more effective in treating humans.

compound

Disclosure of the Invention The present disclosure relates to exemplary immunoadjuvant compounds of formula I or pharmaceutically acceptable salts thereof:

Formula I

Wherein R &lt; ¹ &gt; is -OH or halogen; R ² is -OH, hydrogen or halogen; R &lt; ³ &gt; is hydrogen or halogen; R ⁴ and R ⁵ are each independently selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, Substituted heteroaryl, optionally substituted alkoxy, optionally substituted amino group, or optionally substituted acyl; n is an integer of 1 to 15, and m is an integer of 1 to 20.

In some aspects of the compounds of formula I, R ² is -OH. In some embodiments of compounds of formula I, R &lt; ² &gt; is hydrogen. In some embodiments of the compounds of formula I, R &lt; ² &gt; is halogen. In some embodiments of the compounds of formula I, R ² is F. In some embodiments of compounds of formula I, R &lt; ² &gt; is Cl. In some embodiments of compounds of formula I, R &lt; ² &gt; is Br. In some embodiments of the compounds of formula I, R &lt; ² &gt;

In some embodiments of the compounds of formula I, R &lt; ¹ &gt; is -OH. In some aspects of the compounds of formula I, R &lt; ¹ &gt; is halogen. In some embodiments of the compounds of formula I, R &lt; ¹ &gt; In some embodiments of the compounds of formula I, R &lt; ¹ &gt; is Cl. In some embodiments of the compounds of formula (I), R ¹ is Br. In some embodiments of the compounds of formula I, R &lt; ¹ &gt;

In some embodiments of the compounds of formula I, R &lt; ³ &gt; is hydrogen. In some embodiments of the compounds of formula I, R &lt; ³ &gt; is halogen. In some embodiments of the compounds of formula I, R &lt; ³ &gt; In some aspects of the compounds of formula I, R &lt; ³ &gt; is Cl. In some aspects of the compounds of formula I, R &lt; ³ &gt; is Br. In some embodiments of compounds of formula I, R &lt; ³ &gt;

In some embodiments of compounds of formula I, R &lt; ¹ &gt; is -OH; R ² is -OH, hydrogen or halogen; R &lt; ³ &gt; is hydrogen or halogen. In some embodiments of compounds of formula I, R &lt; ¹ &gt; is -OH; R ² is -OH; R &lt; ³ &gt; is hydrogen or halogen. In some embodiments of compounds of formula I, R &lt; ¹ &gt; is -OH; R ² is hydrogen; R &lt; ³ &gt; is hydrogen or halogen. In some embodiments of compounds of formula I, R &lt; ¹ &gt; is -OH; R ² is halogen; R &lt; ³ &gt; is hydrogen or halogen. In some embodiments of the compounds of formula I, R &lt; ¹ &gt; is halogen; R ² is -OH, hydrogen or halogen; R &lt; ³ &gt; is hydrogen or halogen. In some embodiments of the compounds of formula I, R &lt; ¹ &gt; is halogen; R ² is -OH; R &lt; ³ &gt; is hydrogen or halogen. In some embodiments of the compounds of formula I, R &lt; ¹ &gt; is halogen; R ² is hydrogen; R &lt; ³ &gt; is hydrogen or halogen. In some embodiments of the compounds of formula I, R &lt; ¹ &gt; is halogen; R ² is halogen; R &lt; ³ &gt; is hydrogen or halogen.

In some aspects of the compounds of formula I, R &lt; ⁴ &gt; is phenyl. In some embodiments, R &lt; ⁴ &gt; is an optionally substituted phenyl of formula II:

(II)

특정 양태에서, i는 2이고, R⁴는 하기 화학식 중 하나이다:

특정 양태에서, i는 3이고, R⁴는 하기 화학식 중 하나이다:

특정 양태에서, i는 4이고, R⁴는 하기 화학식 중 하나이다:

특정 양태에서, i는 5이고, R4는 하기 화학식이다:

Wherein i is 0, 1, 2, 3, 4, or 5; When each of R ⁶ is independently hydrogen, _{halogen, -CN, -N0 2, -N 3} , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted Substituted phenyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted amino group, or optionally substituted acyl. In certain embodiments, i is zero. In certain embodiments, i is 1. In certain embodiments, i is 2. In certain embodiments, i is 3. In certain embodiments, i is 4. In certain embodiments, i is 5. In certain embodiments, i is 1 and R &lt; ⁶ &gt; is halogen. In certain embodiments, i is 1 and R &lt; ⁴ &gt; is one of the following formulas:

In certain embodiments, i is 2 and R &lt; ⁴ &gt; is one of the following formulas:

In certain embodiments, i is 3 and R &lt; ⁴ &gt; is one of the following formulas:

In a particular embodiment, and i is 4, R ⁴ is one of the formula:

In certain embodiments, i is 5 and R4 is &lt; RTI ID = 0.0 &gt;

In some embodiments of the compounds of formula I, R &lt; ⁶ &gt; is halogen. In some embodiments of the compounds of formula I, R &lt; ⁶ &gt; In some embodiments of the compounds of formula I, R &lt; ⁶ &gt; is Cl. In some embodiments of compounds of formula I, R &lt; ⁶ &gt; is Br. In some embodiments of compounds of formula I, R &lt; ⁶ &gt;

In some embodiments of compounds of formula I, R &lt; ⁴ &gt; is optionally substituted aryl. In some embodiments of compounds of formula I, R &lt; ⁴ &gt; is a compound of formula III:

(III)

특정 양태에서, k는 2이고, R⁴는 하기 화학식 중 하나이다:

Wherein j is 0, 1, 2, 3, or 4; k is 0, 1, 2, 3, 4, or 5; R ⁷ and R ⁸ is independently selected from the group consisting of hydrogen, halogen, -CN, -NO ₂ , -N ₃ , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, Optionally substituted heteroaryl, optionally substituted heteroaryl, optionally substituted heteroaryl, optionally substituted heteroaryl, optionally substituted heteroaryl, optionally substituted heteroaryl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted amino group, or optionally substituted acyl. In certain embodiments, k is zero. In certain embodiments, k is 1. In certain embodiments, k is 2. In certain embodiments, k is 3. In certain embodiments, k is 4. In certain embodiments, k is 5. In certain embodiments, k is 1 and R &lt; ⁸ &gt; is halogen. In certain embodiments, k is 1 and R &lt; ⁴ &gt; is one of the following formulas:

In certain embodiments, k is 2 and R &lt; ⁴ &gt; is one of the following formulas:

특정 양태에서, k는 3이고, R⁴는 하기 화학식 중 하나이다:

In certain embodiments, k is 3 and R &lt; ⁴ &gt; is one of the following formulas:

특정 양태에서, k는 4이고, R⁴는 하기 화학식 중 하나이다:

In certain embodiments, k is 4, R ⁴ is one of the formula:

특정 양태에서, k는 5이고, R⁴는 하기 화학식이다:

In certain embodiments, k is 5 and R &lt; ⁴ &gt; is of the formula:

In some embodiments of compounds of formula I, n is an integer from 1 to 15 inclusive. In some embodiments of compounds of formula I, n is an integer from 5 to 15. In some embodiments of compounds of formula I, n is an integer from 10 to 15. In some embodiments of the compounds of formula I, n is 10. In some embodiments of the compounds of formula I, n is 11. In some embodiments of the compounds of formula I, n is 12. In some embodiments of the compounds of formula I, n is 13. In some embodiments of compounds of formula I, n is 14. In some embodiments of the compounds of formula I, n is 15.

In some embodiments of compounds of formula I, m is an integer from 1 to 20 inclusive. In some embodiments of the compounds of formula I, m is an integer from 5 to 15. In some embodiments of the compounds of formula I, m is an integer from 5 to 15. In some embodiments of the compounds of formula I, m is an integer from 10 to 15. In some embodiments of compounds of formula I, m is 10. In some embodiments of the compounds of formula I, m is 11. In some embodiments of the compounds of formula I, m is 12. In some embodiments of the compounds of formula I, m is 13. In some embodiments of the compounds of formula I, m is 14. In some embodiments of the compounds of formula I, m is 15.

In some aspects of the compounds of formula I, R &lt; ⁷ &gt; is hydrogen; R ⁸ is F; k is 1, 2 or 3; In some aspects of the compounds of formula I, R ⁷ is F; R ⁸ is hydrogen; j is 1, 2 or 3; In some embodiments of the compounds of formula I, R ⁷ and R ⁸ are both F; k is 1, 2 or 3; j is 1, 2 or 3;

In some embodiments of the compounds of formula I, the provided compounds are one of the following compounds:

Pharmaceutical composition

The disclosure of the present invention provides pharmaceutical compositions comprising the exemplary compounds described herein and pharmaceutically acceptable excipients. The compositions disclosed herein may be included in a pharmaceutical or functional food composition with additional active agents, carriers, vehicles, excipients or adjuvants which may be ascertained by one skilled in the art upon reading the disclosure of the present disclosure.

The pharmaceutical composition preferably comprises one or more pharmaceutically acceptable carriers. In such pharmaceutical compositions, the compositions disclosed herein also form "active compounds" which are referred to as "active agents. &Quot; The term " pharmaceutically acceptable carrier " as used herein includes solvents, dispersion media, coatings, antibacterial and antifungal agents compatible with pharmaceutical administration, isotonic and absorption delaying agents and the like. Supplementary active compounds may also be incorporated into the compositions. The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, for example, intravenous, transdermal, subcutaneous, oral (e.g., inhaled), transdermal (topical), transmucosal and rectal administration. Solvents or suspensions used for parenteral, intradermal or subcutaneous applications may include the following components: a sterile diluent, for example, water for injection, saline solution, nonvolatile oil, polyethylene glycol, glycerin, propylene glycol, Or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium hydrogen sulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers, such as, for example, acetate, citrate, or phosphate, and agents for tonicity adjustment such as sodium chloride or dextrose. The pH can be adjusted to an acid or base, for example, hydrochloric acid or sodium hydroxide. Parenteral formulations may be included in ampules, disposable syringes or multi-dose vials made of glass or plastic.

Objects such as those used herein include humans and non-human primates (e.g., guerrillas, macaques, marmosets), livestock animals (e.g., sheep, cows, horses, donkeys and pigs), pets For example, dogs, cats), experimental test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g. foxes, deer) Refers to any other organism capable of crying. There is no restriction on the type of animal that can benefit from the presently described formulation. A subject, whether it is a human or a non-human organism, can be referred to as a patient, an individual, an animal, a host, or a recipient.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersing agents and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy injectability exists. Must be stable under the conditions of manufacture and storage and must be preserved from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it may be desirable to include isotonic agents, for example, sugars, polyhydric alcohols, such as mannitol, sorbitol or sodium chloride, in the composition. The delayed uptake of the injectable composition can be caused by including agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

The use of the compositions described herein

The invention provides a composition useful for stimulating an immune response in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition as disclosed herein.

The compositions described herein can also be used to raise the production of invariant natural killer T (iNKT) cells in a human subject in need thereof, which method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutically acceptable composition, Wherein the composition comprises the exemplary compounds described herein.

The exemplary compositions described herein may also be used to stimulate the production of cytokines and / or chemokines in a human subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition, , Wherein the composition comprises an amount sufficient to increase the cytokine / chemokine production of the compounds described herein.

"Effective" amount or "therapeutically effective amount" of an active agent means an amount of agent that is non-toxic and sufficient to provide a beneficial effect. The "effective" amount of active agent varies from subject to subject depending on the age and general condition of the subject, the particular active agent or active agents, and the like. Unless otherwise indicated, the term "therapeutically effective" amount as used herein is intended to encompass an amount effective for treatment of adverse conditions as well as an amount effective for the prevention of adverse conditions and / or alleviation of adverse conditions.

A therapeutically effective amount of the active compound (i. E., Effective dose) as defined herein may range from about 0.001 to 100 g / kg body weight, or may be other ranges that will be apparent to those skilled in the art without undue experimentation. Those skilled in the art will appreciate that certain factors can affect the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatment, general health or age of the subject, Do not.

The adverse condition as the term is used herein may be a "normal" condition commonly seen in an individual or may be a pathological condition that may or may not be associated with a named disease.

The term "lipid " as used herein refers to any lipophilic (lipophilic) molecule that participates in the cellular signaling pathway.

The term "glycolipid " as used herein refers to a carbohydrate-attached lipid that acts as a marker for cell recognition.

In accordance with another aspect, one or more sub-kits may be considered by those skilled in the art, and the sub-kit may perform at least one of the methods disclosed herein, wherein the sub-kit comprises two or more compositions, Or in combination with an effective amount of a composition disclosed herein according to one or more of the above-mentioned methods.

The kit also includes a composition comprising an active agent, a determinant of a biological event or other compound that can be ascertained by one skilled in the art upon reading the disclosure of the present invention. The kit may also comprise at least one composition comprising an effective amount of the composition or cell line disclosed herein. The composition and cell line of the sub-kit are used to carry out at least one of the methods disclosed herein according to procedures that can be ascertained by those skilled in the art.

The term "specifically binding" as used herein refers to the interaction between a binding pair (e. G., An antibody and an antigen). In various cases, are specifically binding may be implemented by about ^10-6 mol / L, about 10 ^{to 7} mole / liter, or an affinity of about 10 ^-8 mole / L or less constant.

It will be apparent to those skilled in the art upon reading this disclosure that each of the individual aspects described and described herein can be readily separated and combined from any other separate embodiment without departing from the scope or spirit of the present invention. Individual components and features. Any recited method may be performed in the order of the events mentioned or in any other order possibly legitimately.

In one aspect, the immunogenic compositions described herein may be administered parenterally (e.g., by intravenous, subcutaneous, or intramuscular injection). Alternatively, other modes of administration, including suppositories and oral formulations, may be preferred. For suppositories, binders and carriers may include, for example, polyalkylene glycols or triglycerides. Oral formulations may include, for example, commonly used ingredients such as pharmaceutical grade saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of a solvent, suspension, tablet, capsule, delayed-release formulation or powder and contain from 10 to 95% of the immunogenic composition described herein.

The immunogenic composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective, protective and immunogenic amount. The amount administered will depend, for example, on the subject to be treated, including the ability of the individual immune system to synthesize antibodies and, if desired, to produce a cell-mediated immune response. The exact amount of active ingredient to be administered depends on the judgment of the practitioner. However, suitable dosage ranges are readily determined by those skilled in the art. Therapies suitable for initial administration and booster doses may also vary, but may include initial administration followed by subsequent administration. The dose of the vaccine may also depend on the route of administration and will vary with the size of the host.

The immunogenic compositions of the invention can also be used to produce antibodies in animals for the production of antibodies that can be used in both cancer therapy and diagnosis. Methods for producing monoclonal and polyclonal antibodies and fragments thereof in animals (e. G., Mouse, rabbit, goat, sheep or horse) are well known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The term "antibody" refers to a whole immunoglobulin such as Fab, F (ab ') ₂ , Fv, scFv (single chain antibody), and dAb (domain antibody; Ward, et al. (1989) Nature, 341, 544) Molecules as well as fragments thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of a dispute, this document containing definitions will be adjusted. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes, including describing and describing the chemicals, cell lines, vectors, animals, devices, statistical analyzes and methods reported in the publications that may be used in connection with the present invention Quot; is hereby incorporated by reference. All references cited herein are to be construed as indicating a level in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by a prior invention.

Before the materials and methods of the present disclosure are described, the present invention is not limited to the particular methods, protocols, materials, and reagents described, as they may vary. It is also to be understood that the technical terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is to be limited only by the claims.

Where a range of values is provided, each intervening value is between the upper and lower limits of the range, unless expressly indicated otherwise, and any other stated or intervening value in the stated ranges, Are understood to be included. The upper and lower limits of these smaller ranges may independently be included in a smaller range and apply to any specifically excluded range included in the invention and also in the stated range. Where the stated range includes one or both of the limits, the scope excluding one or both of the above is included.

Example

The following examples are presented to enable those skilled in the art to provide a description of the entire invention and methods of manufacture and use according to the invention and are not intended to limit the scope of what the inventors regard as their discoveries. Although efforts have been made to ensure accuracy with respect to the number used (eg, quantity, temperature, etc.), some experimental errors and deviations should be considered. Unless otherwise indicated, parts and parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, pressure is at or near atmospheric.

General : All reagent chemicals were purchased at reagent grade and used without further purification. Anhydrous solvents such as dichloromethane (CH ₂ Cl ₂ ), tetrahydrofuran (THF), N, N-dimethylformamide (DMF), methanol (MeOH) and pyridine were purchased from Acros. HPLC grade solvent chloroform (CHCl ₃ ) and methanol were purchased from Merck. The molecular sieve 4 A (MS 4 A) for glycosylation was purchased from Acros and activated by firing. The reaction was visualized by analytical thin layer chromatography (TLC) on EM silica gel 60 F254 plates and staining with UV (254 nm) and / or with acidic seric ammonium molybdate or ninhydrin. Flash column chromatography was performed on silica gel 60 Geduran (40-63 [mu] m, Merck). Biogel LH20 for purification of the final product was purchased from Aldrich. ¹ H NMR spectra were recorded on a Bruker Topspin-600 (600 MHz) spectrometer at 20 ° C. Chemical shifts (δ ppm) were assigned according to internal standard signals of CDCl ₃ (δ = 7.24 ppm), MeOD (δ = 3.31 ppm), and pyridine-d ⁵ (δ = 7.58 ppm). ¹³ C NMR spectra were obtained on a Bruker Topspin-600 (150 MHz) spectrometer and recorded for δ ppm scale using a signal of CDCl ₃ (δ = 77.23 ppm), MeOD (δ = 49.15 ppm) for calibration. The coupling constant ( J ) was recorded in Hz. The splitting pattern is described using the following abbreviations: s, single line; d, doublet; t, triple line; dd, double doublet; m, multiplet. ¹ H NMR spectra are recorded in this order: chemical shift; Multiplicity; Number (s) of protons; Coupling constant (s).

Chemical synthesis

Glucosyl  Donor 8 Synthesis of

Compound 2 : To a solution of 1,2,3,4,6-penta-O-acetyl -? - D-glucopyranose 1 (40 g, 102.5 mmol) in 200 mL anhydrous CH ₂ Cl ₂ was added p- Thiol (15.4 g, 123 mmol) and BF ₃ OEt ₂ (15.4 mL, 123 mmol) were added and the reaction stirred at ambient temperature for 16 hours under argon. The resulting solution was saturated NaHC0 ₃ solution and extracted directly with brine, dried with MgS0 ₄ and evaporated. It was then recrystallized in AcOEt-hexane solution to give 2 as a white solid (32.6 g, 70%).

Compound 3 : A catalytic amount of sodium methoxide (NaOMe) was added to a solution of 2 (32.6 g, 71.8 mmol) in 500 mL of anhydrous MeOH and stirred at ambient temperature for 3 hours. The reaction was neutralized by addition of Amberlite IR-120, filtered and the resulting solution was concentrated to dryness to give 3 (20.3 g, 99%) as a white solid which was used directly in the next reaction without further purification.

Compound 4 : To a solution of 3 (11.1 g, 38.8 mmol) in 48 mL of anhydrous pyridine was added triphenylmethyl chloride (13.5 g, 46.6 mmol). The reaction was stirred at 60 &lt; 0 &gt; C under argon for 16 hours. After removal of the solvent, the mixture was purified by flash column chromatography on silica gel (hexane: AcOEt: MeOH 1: 1: 0.1) to give 4 (12.3 g, 60%) as a white powder.

Compound 5 : To a solution of 4 (21.1 g, 39.9 mmol) in 200 mL of anhydrous N, N-dimethylformamide (DMF) was added sodium hydride (60% in mineral oil) (5.8 g, 143.6 mmol) at 0 ° C. The reaction was stirred for 1 hour, then benzyl bromide (17.2 mL, 143.6 mmol) was added and stirred at ambient temperature for 16 hours under argon. The reaction was quenched with MeOH and evaporated to dryness. The residue was diluted with AcOEt, and the solution was washed with H ₂ 0 and brine, dried over MgS0 _4, it evaporated to dryness. The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 9: 1) to give 5 (22.3 g, 70%) as a white powder.

Compound 6 : To a solution of 5 (30.0 g, 37.5 mmol) in 1065 mL of an aqueous acetic acid solution (AcOH: H ₂ O 4: 1) was stirred at 75 ° C for 3 hours. After removal of the solvent, the residue was purified by flash column chromatography on silica gel (hexane: AcOEt 2: 1) to give 6 (16.7 g, 80%) as a white solid.

Compound 7 : To a solution of 6 (5.0 g, 9.0 mmol) in 18 mL of anhydrous pyridine was added acetic anhydride (1.0 mL). The reaction was stirred under argon at ambient temperature for 16 hours. After removal of solvent, the residue was diluted with AcOEt, and the solution was washed with H ₂ 0 and brine, dried over MgS0 _4, evaporated to dryness. The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 5: 1) to give 7 (5.3 g, 99%) as a white solid.

Compound 8 : N-Bromosuccinimide (1.7 g, 9.5 mmol) was added to a solution of 7 (5.5 g, 9.2 mmol) in 129 mL of an acetone aqueous solution (acetone: H ₂ O 4: The reaction was stirred at ambient temperature for 1 hour. After removal of the solvent, the residue was diluted with AcOEt, extracted with H ₂ O, aqueous sodium thiosulfate (NaS ₂ O ₃ ) solution, brine, and dried over MgSO ₄ . The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 2: 1) to provide 8 (3.1 g, 69%, α / β = 1: 1) as a white solid.

Receptor 18 Synthesis of

Compound 13 : To a solution of D-Lyxose 12 (20 g, 133 mmol) in anhydrous N, N-dimethylformamide (DMF) was added 2-methoxypropene (15 mL, 160 mmol) and camphor-10- CSA) (3 g, 13.3 mmol). The reaction was stirred under argon at ambient temperature for 16 hours. The solution was quenched with triethylamine (Et ₃ N), evaporated to dryness and purified directly by flash column chromatography on silica gel (hexane: AcOEt: MeOH 1: 1: 0.2) to afford 13 (21 g, 83% As a solid.

Compound 14 : Triphenylmethyl chloride (37.8 g, 132 mmol) was added to a stirred solution of 13 (21 g, 110 mmol) in 140 mL of anhydrous pyridine. The reaction was stirred at 60 &lt; 0 &gt; C under argon for 16 hours. The solution was concentrated to dryness, the residue dissolved in ethyl acetate (AcOEt) and, H ₂ 0, evaporated was washed with brine, dried over magnesium sulfate (MgSO _4), then. The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 1: 2) to give 14 (36.5 g, 77%) as a white powder.

Compound 15 : Lithium bis (trimethylsilyl) amide (LHMDS) (20 mL of a 1 M solution in THF, 20 mmol) was added to a stirred solution of 14 (8.4 g, 19.4 mmol) in 40 mL anhydrous tetrahydrofuran (THF) And the reaction was stirred under argon for 1 hour. It was refluxed in toluene for 5 days in dry THF 83mL of 1- bromo-tridecane (C ₁₃ H ₂₇ Br) and triphenylphosphine (PPh ₃₎ above prepared from the TIG reagent _{(Wittig reagent) C 13 H 27} PPh 3 LHMDS (40 mL of 1M solution in THF, 40 mmol) was added at 0 [deg.] C to a stirred solution of Br (20.1 g, 38.2 mmol) and the reaction stirred under argon for 1 hour to give a pale orange yildide. A solution of 14 was added dropwise to the iodide at 0 &lt; 0 &gt; C, the reaction was allowed to warm to ambient temperature and stirred under argon for 9 hours. The resulting solution was quenched with MeOH and evaporated to dryness. The residue was diluted with AcOEt and extracted with H ₂ 0 and brine, dried over MgS0 _4, then concentrated. The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 15: 1) to give 15 (8.7 g, 75%) as a colorless oil.

Compound 16 : Compound 16 was prepared from 15 (1 g, 1.7 mmol) catalytic hydrogenation in 10 mL anhydrous MeOH containing a catalytic amount of palladium hydroxide on carbon (20% Pd). The suspension was stirred in H ₂ atmosphere for 4 hours. The solution was filtered through Celite 545 to remove the catalyst, evaporated to dryness and then purified by flash column chromatography on silica gel (hexane: AcOEt 20: 1) to afford 16 (903 mg, 90%) as a white solid Lt; / RTI &gt;

Compound 17 : 2,6-Lutidine (3.5 mL, 30 mmol) was added to a solution of 16 (5 g, 8.3 mmol) and 4 A molecular sieve (1 g) in 39 mL anhydrous CH ₂ O ₂ at ambient temperature. The solution was cooled to -45 ℃, was added dropwise trifluoroacetic methanesulfonic anhydride _{(Tf 2 0) (2.67mL,} 15.9mmol) , stirred for 1 hour the reaction under argon. Tetramethylguinidinium azide (TMGA) (3.9 g, 25 mmol) was then added and the reaction was warmed to ambient temperature and stirred under argon for 16 h. The resulting solution was filtered through Celite 545 to remove the 4 A molecular sieve, the residue was diluted with CH ₂ Cl ₂ , extracted with H ₂ O and brine, dried over MgSO ₄ and evaporated. The mixture was simply purified by flash column chromatography on silica gel (hexane: AcOEt 20: 1) to remove most of the impurities and used directly in the next step.

Compound 18 : To a solution of 17 in 20 mL of anhydrous CH ₂ Cl ₂ was added tetrafluoroacetic acid / tetrafluoroacetic anhydride (TFA / TFAA 1.8 M / 1.8 M in CH ₂ Cl ₂ ) (14 mL, 24.9 mmol) Was added and stirred under argon for 15 minutes. The reaction was quenched by the addition of 10 mL of Et ₃ N, then poured into 200 mL of methanol (MeOH) and stirred for an additional 15 minutes. After removal of the solvent, the residue was diluted with AcOEt, extracted with H ₂ O, saturated NaHCO ₃ solution and brine, and dried over MgSO ₄ . The organic layer was concentrated in vacuo and the mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 10: 1) to give 18 (2 g, 2 step 63%) as a yellow oil.

α- Glucosylceramide  Analog 24-26 Synthesis of

Compound 19 : Glucosyl Donor 8 (2.9 g, 5.9 mmol), dimethylsulfide (590,, 7.8 mmol), 4 Å molecular sieve (500 mg) and 2-chloropyridine (1.8 mL, 19.5 mmol) in anhydrous CH ₂ Cl ₂ mmol) in dichloromethane (5 mL) was added trifluoromethanesulfonic anhydride (1 mL, 6 mmol) at -45 &lt; 0 &gt; C under argon. The reaction at -45 ℃ for 20 minutes, in a 0 ℃ for 20 min and ambient temperature and stirred for 20 minutes more was added and then the acceptor 18 (1.5g, 3.9mmol) in 5mL of CH ₂ Cl _2. The reaction was stirred under argon at ambient temperature for 16 hours. The solution was filtered through Celite 545 to remove the molecular sieve. After removal of solvent, the residue was diluted with AcOEt, and the solution was washed with H ₂ 0 and brine, dried over MgS0 _4, evaporated to dryness. The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 10: 1) to give 19 as a colorless oil (2 g, 60%).

Compound 20: pyridine / H ₂ 0: 19, triphenylphosphine (165mg, 0.63mmol) to a solution of (269mg, 0.31mmol) of (10 1, 12mL) was added. The reaction was stirred at 45 &lt; 0 &gt; C under argon for 16 hours. After removal of solvent, the residue was diluted with AcOEt, H ₂ was washed, dried over MgS0 ₄ to 0 and brine and dried then evaporated. The mixture was used in the next step without further purification.

Compound 21 : Hexacosanoic acid (159 mg, 0.4 mmol), EtN (88 [mu] l), EDC (90 mg, 0.47 mmol) and HBTu (178 mg, 0.47 mmol) were added to a solution of 20 in 36 mL anhydrous CH ₂ Cl ₂ . The reaction was stirred under argon at ambient temperature for 16 hours. After removal of solvent, the residue was diluted with AcOEt, H ₂ 0, dried was washed with brine, dried over MgS0 ₄ and then evaporated. The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 4: 1) to provide 21 as a white solid (293 mg, 78%, step 2).

Compound 22 : Sodium methoxide (0.024 mmol) was added to a solution of 21 (293 mg, 0.24 mmol) in 50 mL of co-solvent (MeOH: CH ₂ Cl ₂ 1: 1) and stirred at ambient temperature for 6 h under argon . The reaction was neutralized with Amberlite IR-120 and filtered. After removal of the solvent, the residue was used in the next step without further purification.

Compound 23 : The hydrolyzed compound 22 was dissolved in 50 mL of an aqueous acetic acid solution (AcOH: H ₂ O 4: 1) and stirred at 60 ° C for 16 hours. After removal of the solvent, the mixture was purified by flash column chromatography on silica gel (hexane: AcOEt: MeOH 1: 1: 0.1).

Compound 24

The deacetonide derivative 23 was dissolved in 50 mL of co-solvent (MeOH: CHCl ₃ 4: 1) containing palladium hydroxide / carbon (20% Pd) (cat.) And stirred in H ₂ atmosphere for 16 hours. The solution was filtered through Celite 545 to remove the catalyst and evaporated to dryness, and the mixture on a silica gel flash column chromatography _{(MeOH: CHCl 3 1: 9} ) was purified by and _{LH 2 0 (MeOH: CHC1 3} 1: 1 ) To provide 24 (72 mg, 5% for 3 steps) as a white solid.

Compound 25

Compound 25 was synthesized using a procedure analogous to compound 24 .

Compound 26

Compound 26 was synthesized using a procedure analogous to compound 24 .

Fluorinated donor 38 Synthesis of

Compound 32 : To a solution of 1,2,3,4,6-penta-O-acetyl -? - D-glucopyranose 1 in anhydrous CH ₂ Cl ₂ was added 4-methoxyphenol and BF ₃ OEt ₂ And the reaction was stirred at ambient temperature under argon for 16 hours. The resulting solution was saturated NaHC0 ₃ solution and extracted directly with brine, dried with MgS0 ₄ and evaporated. The product was recrystallized from a solution of AcOEt-hexane to provide 32 as a white solid.

Compound 33 : A catalytic amount of sodium methoxide (NaOMe) was added to a solution of 32 in dry MeOH and stirred at ambient temperature for 3 hours. The reaction was neutralized by addition of Amberlite IR-120, filtered and the resulting solution was concentrated to dryness to afford 33 as a white solid which was used directly in the next step without further purification.

Compound 34: Anhydrous cosolvent benzaldehyde dimethyl acetal and sodium methoxide (NaOMe) to a catalytic amount of a solution of 33 (DMF and CH ₃ CN) was added. The reaction was stirred at ambient temperature for 16 hours. The solution was neutralized by addition of Et ₃ N and concentrated. The mixture was taken up in ethyl acetate, washed with saturated NaHC0 ₃ solution and brine, dried over MgS0 ₄ and evaporated. The product was recrystallized from a solution of AcOEt-hexane to afford 34 as a white solid.

Compound 35 : Sodium hydride (60% in mineral oil) was added at 0 C to a solution of 34 in anhydrous N, N-dimethylformamide (DMF). The reaction was stirred for 1 hour, then benzyl bromide was added and the reaction was stirred at ambient temperature for 16 hours under argon. The solution was quenched with MeOH and evaporated to dryness. The residue was diluted with AcOEt, yongaekreul H ₂ 0 and washed with brine, dried over MgS0 _4, it evaporated to dryness. The product was recrystallized from a solution of AcOEt-hexane to provide 35 as a white solid.

Compound 36 : To a solution of 35 in CH ₂ Cl ₂ was added triethylsilane and trifluoroacetic acid (TFA) at 0 ° C. The reaction was stirred at ambient temperature for 3 hours. The solution was washed directly with H ₂ O, saturated NaHCO ₃ solution and brine, dried over MgSO ₄ and evaporated to dryness. The product was recrystallized from a solution of AcOEt-hexane to provide 36 as a white solid.

Compound 37 : To a solution of 36 in CH ₂ Cl ₂ was added diethylaminosulfur trifluoride (DAST). The reaction was stirred at 45 ℃ for 16h, the solution was washed directly with H ₂ 0, saturated NaHC0 ₃ solution and brine, dried over MgS0 ₄ and evaporated to dryness. The residue was purified by flash column chromatography on silica gel to provide 37 as a colorless oil.

Compound 38: the serik ammonium nitrate (CAN) in a solution of 37 in anhydrous co-solvent (toluene, H ₂ 0 and CH ₃ CN) was added. The reaction was stirred at ambient temperature for 10 minutes. The solution was extracted with ethyl acetate, washed with H ₂ O, saturated NaHCO ₃ solution and H ₂ O, dried over MgSO ₄ and evaporated to dryness. The residue was purified by flash column chromatography on silica gel to provide 38 as a colorless oil.

Fluorinated analog 43 Synthesis of

Compound 39 : To a solution of Donor 38 , dimethyl sulfide, 4A molecular sieve and 2-chloropyridine in anhydrous CH ₂ Cl ₂ was added trifluoromethanesulfonic anhydride at -45 ° C under argon. The reaction was stirred at -45 ℃ for 20 minutes, and stirred at 0 ℃ for 20 minutes, stirred for 20 minutes more at ambient temperature, followed by addition of the receptor 18 in CH ₂ Cl _2. The reaction was stirred under argon at ambient temperature for 16 hours. The solution was filtered through Celite 545 to remove the molecular sieve. After removal of the solvent, the residue was diluted with AcOEt and the solution was washed with H ₂ O and brine, dried over MgSO ₄ and evaporated to dryness. The mixture was purified by flash column chromatography on silica gel to give 39 .

Compound 40: pyridine / H ₂ O: 39 To a solution of triphenylphosphine in toluene (10 1) was added. The reaction was stirred at 45 &lt; 0 &gt; C under argon for 16 hours. After removal of the solvent, the residue was diluted with AcOEt, extracted with H ₂ O, brine, dried over MgSO ₄ and evaporated to dryness. The mixture was used in the next step without further purification.

Compound 41 : To a solution of 40 in anhydrous CH ₂ Cl ₂ was added 4- (4-fluorophenoxy) phenyl undecanoic acid, Et ₃ N, EDC and HBTu. The reaction was stirred under argon at ambient temperature for 16 hours. After removal of solvent, the residue was diluted with AcOEt, H ₂ 0, was extracted with brine, dried, and then dried over MgS0 _4, and evaporated. The mixture was purified by flash column chromatography on silica gel to afford 41 .

Compound 42 : To a solution of compound 41 in acetic acid aqueous solution (AcOH: H ₂ O 4: 1) was stirred at 60 ° C for 16 hours. After removal of the solvent, the mixture was dissolved in ethyl acetate and washed with saturated NaHCO ₃ solution and brine, dried over MgSO ₄ and evaporated. The residue was purified by flash column chromatography on silica gel to give 42 .

Compound 43

Acetonide derivative having 42 to a co-solvent containing palladium hydroxide / carbon (20% Pd) (cat. ) Was dissolved in _{(MeOH:: CHC1 3 4 1} ), and the mixture was stirred for 16 hours in a H ₂ atmosphere. The solution was filtered through celite 545 to remove the catalyst, evaporated to dryness, and the mixture was purified by flash column chromatography on silica gel, eluting with LH ₂ O to give 43 .

Fluorinated donor 48 Synthesis of

Compound 44 : To a solution of 32 in anhydrous pyridine was added triphenylmethyl chloride. The reaction was stirred at 60 &lt; 0 &gt; C under argon for 16 hours. After removal of the solvent, the mixture was purified by flash column chromatography on silica gel to afford 44 .

Compound 45 : Sodium hydride (60% in mineral oil) was added at 0 C to a solution of 44 in N, N-dimethylformamide (DMF). The reaction was stirred for 1 hour, then benzyl bromide was added and stirred at ambient temperature for 16 hours under argon. The reaction was quenched with MeOH and evaporated to dryness. The residue was diluted with AcOEt, and the solution was washed with H ₂ 0 and brine, dried over MgS0 _4, it evaporated to dryness. The mixture was purified by flash column chromatography on silica gel to give 45 .

Compound 46 : To a solution of 45 in aq. Acetic acid (AcOH: H ₂ O 4: 1) was stirred at 75 ° C for 3 hours. After removal of solvent, the mixture was diluted with AcOEt, and the solution was washed with H ₂ 0 and brine, dried over MgS0 _4, evaporated to dryness. The residue was purified by flash column chromatography on silica gel to give 46 .

Compound 47 : Diethylaminosulfurtrifluoride (DAST) was added to a solution of 46 in CH ₂ Cl ₂ . The reaction was stirred at 45 ℃ for 16h, the solution was washed with H ₂ 0, saturated NaHC0 ₃ solution and brine, dried over MgS0 ₄ and evaporated to dryness. The residue was purified by flash column chromatography on silica gel to give the compound.

Compound 48: the serik ammonium nitrate (CAN) in a solution of 47 in anhydrous co-solvent (toluene, H ₂ 0 and CH ₃ CN) was added. The reaction was stirred at ambient temperature for 10 minutes. The solution was extracted with ethyl acetate, washed with H ₂ O, saturated NaHCO ₃ solution and H ₂ O, dried over MgSO ₄ and evaporated to dryness. The residue was purified by flash column chromatography on silica gel to afford 48 as a colorless oil.

Fluorinated analog 53 Synthesis of

Compound 49 : To a solution of Donor 48 , dimethyl sulfide, 4A molecular sieve and 2-chloropyridine in anhydrous CH ₂ Cl ₂ was added trifluoromethanesulfonic anhydride at -45 ° C under argon. The reaction was stirred at -45 ℃ for 20 minutes, and stirred at 0 ℃ for 20 minutes, stirred for 20 minutes more at ambient temperature, followed by addition of the receptor 18 in CH ₂ Cl _2. The reaction was stirred under argon at ambient temperature for 16 hours. The solution was filtered through Celite 545 to remove the molecular sieve. After removal of the solvent, the residue was diluted with AcOEt and the solution was washed with H ₂ O and brine, dried over MgSO ₄ and evaporated to dryness. The mixture was purified by flash column chromatography on silica gel (hexane: AcOEt 10: 1) to give 49 .

Compound 50: pyridine / H ₂ 0: 49 To a solution of triphenylphosphine in toluene (10 1) was added. The reaction was stirred at 45 &lt; 0 &gt; C under argon for 16 hours. After removal of solvent, the residue was diluted with AcOEt, H ₂ 0, was extracted with brine, dried, and then dried over MgS0 _4, and evaporated. The mixture was used in the next step without further purification.

Compound 51 : To a solution of compound 50 in anhydrous CH ₂ Cl ₂ was added 4- (4-fluorophenoxy) phenyl undecanoic acid, Et ₃ N, EDC and HBTu. The reaction was stirred under argon at ambient temperature for 16 hours. After removal of solvent, the mixture was diluted with AcOEt, H ₂ 0, dried was extracted with brine, dried over MgS0 ₄ and then evaporated. The residue was purified by flash column chromatography on silica gel to afford 51 .

Compound 52 : A solution of Compound 51 was dissolved in an aqueous acetic acid solution (AcOH: H ₂ O 4: 1) and stirred at 60 ° C for 16 hours. After removal of solvent, the mixture was diluted with AcOEt, H ₂ 0, dried was extracted with brine, dried over MgS0 ₄ and then evaporated. The residue was purified by flash column chromatography on silica gel.

Compound 53

Acetonitrile derivatives having the cyanide 52 cosolvent containing palladium hydroxide / carbon (20% Pd) (cat. ) Was dissolved in _{(MeOH:: CHC1 3 4 1} ), and the mixture was stirred for 16 hours in a H ₂ atmosphere. The solution was filtered through celite 545 to remove the catalyst, evaporated to dryness, and the mixture was purified by flash column chromatography on silica gel, eluting with LH ₂ O to give 53 .

Biological research

Injection of glycated analogs in mice

All glycolipids were dissolved in 100% DMSO at a concentration of 1-2 mg / ml. For in vivo experiments, all compounds were diluted to 10 占 퐂 / ml in saline immediately before injection into mice with 100 占 퐇 of diluted glycolipids or 100 占 퐇 of 1% DMSO. 6 to 12 weeks of pathogen-free C57BL / 6 female mice were obtained from an institution (National Laboratory Animal Center, Taipei, Taiwan). Jα18 knockout (KO) B6 mice were donated from Masaru Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan). All mice were maintained in the pathogen-free animal cage of the institute (Institute of Cellular and Organismic Biology, Academia Sinica (Taipei, Taiwan)).

Determination of murine cytokine / chemokine secretion

B6 WT or Jα18 KO mice were intravenously injected with vehicle or glycolipid at 0.1 or 1 μg / mouse. Serum was collected at 2 and 18 hours post-injection for the measurement of cytokines / chemokines by Beadlyte® mouse cytokine kit (Millipore, NY) and read by the Luminex® 200 ™ system (Luminex, Austin, TX).

After the specific glycolipid stimulation, FACS  analysis

B6 WT or Jα18 KO mice treated with specific glycolipids (1 μg / mouse) or vehicle (1% DMSO in PBS) were sacrificed at 72 hours post injection and the spleen was collected. After squeezing the spleen through a 70 μm strainer and hydrolyzing the red blood cells, the nucleated cells were resuspended in PBS buffer containing azide (0.05%) and incubated for 30 minutes at 4 ° C. with antibodies Lt; / RTI &gt; After washing, spleen cells were applied to FACS analysis. Antibodies against CD3, CD4, CD8a, CD11c, CD80, and CD86 were obtained from the organ [BD Bioscience-Pharmingen].

mCD1d And the binding strength of the bicomponent complex between glycolipids

Different concentrations of the mCD1d ^di -glycolipid complex coated on ELISA plates were incubated with a saturating amount of L363 antibody conjugated with biotin (BioLegend) followed by streptavidin-HRP detection and ELISA. The KD between the L363 antibody and the designated mCD1d ^di -glycolipid complex was calculated from a linear regression analysis of the scatched transformation of the L363 antibody binding curve using GraphPad prism software. L363 was found to be similar to the bonding strength to recognize mCD1d -7DW8-5 ^di-Glc complex and mCD1d ^d1 -7DW8-5 complex. Next, the KD of the binary complex was determined as follows. Different concentrations of glycolipids were incubated at 37 째 C overnight with a fixed amount of mCD1d dimer and the mCD1d ^di -glycolipid complex was then coated on a 96 well ELISA plate overnight at 4 째 C. After washing and blocking with BSA for 1 hour at room temperature (RT), the biotin conjugated L363 antibody was added at room temperature for 30 minutes, then incubated with streptavidin-HRP for 30 minutes at RT and detected with an ELISA reader . The KD values of the two complexes were calculated from a linear regression analysis of the Scatchard transformation of the L363 antibody binding curve.

human iNKT  Expansion of cells

Human pure Vα24 ⁺ iNKT cells were cultured with autologous immature CD14 ⁺ DC pulsed with either 100 ng / ml or DGLO-specific glycolipids for 18 hours on day 2. On the third day, the suspension cells were transferred to a new dish and cultured in the presence of 50 U / ml IL-2 (R & D Systems) and supplemented with fresh medium every 3 days. Percentage of Vα24 ⁺ / Vβ11 ⁺ cells was determined by flow cytometry on day 9. Total cell counts after expansion were calculated by the Guava ViaCount reagent (Millipore, USA) and detected by the Guava system with CytoSoft software including the ViaCount module (Millipore, USA).

V? 14 + iNKT  A variety of cells CD1d - Loaded  Glycolipid valence

Briefly, a 1:10 molar ratio of carbohydrate or vehicle was loaded in a rat CD1d: Ig dimer (BD Biosciences PharMingen, San Diego, Calif.) Overnight at 37 ° C. Rat 1.2 Vα14 ⁺ iNKT cells were incubated at various concentrations of dimer-glycolipid complexes in buffer containing azide (0.05%) for 30 minutes at 4 ° C. These cells were then stained with anti-mouse IgG1-PE mAb (A85-1) at 4 ° C for another 30 minutes and then washed and fixed with 4% paraformaldehyde (PFA) and the bound CD1d dimer complex Cell counting method. Bounding curves and linear pits of the Scatchard transform were plotted by Graphpad prism software.

Vα24 ⁺ iNKT  With cells CD1d Loaded  Glycolipid valence

The binding of human CD1d-glycolipid complex to V? 24 ⁺ iNKT cells expanded by 7DW8-5 at 100 ng / ml was determined as previously described. ¹⁹

Vα24 ⁺ iNKT  Cell line and Unmature  Isolation and Generation of Monocyte-Derived Dendritic Cells

Vα24 ⁺ iNKT cells and CD14 ⁺ cells were isolated from peripheral blood cells as previously described. ¹⁹ unmatured DCs were generated from CD14 ⁺ cells after 2 days of incubation in the presence of 300 U / ml GM-CSF (R & D Systems) and 100 U / ml IL-4 (R & D Systems).

The Vα24 ⁺ iNKT cell line expanded with 7DW8-5 or CI was generated as follows. After irradiation at 2,000 rad, unmature DCs were co-cultured with homologous Vα24 ⁺ iNKT cells in the presence of 7DW8-5 or CI at 100 ng / ml for 1 day. The cells were expanded in the presence of 50 U / ml IL-2 for 10-14 days after lipolysis. The same procedure was repeated once for additional stimulation and expansion of iNKT cells. The 7DW8-5 or C1-expanded iNKT cell line was shown to express the Vα24 T cell antigen receptor (> 95% purity).

mCD1d  versus hCD1d Swapping  analysis

Rat DN3A4-1.2 Vα14 ⁺ iNKT hybridoma cells or C1-expanded Vα24 ⁺ iNKT cells were assigned to mCD1d (A20-CD1d cells) or hCD1d (HeLa-CD1d cells) at 1, 0.1, and 0.01 μg / And pulsed with glycolipid antigen. After 18 hours, supernatant was collected for determination of cytokine secretion (s). IL-2 released from Vα14 ⁺ iNKT cells was determined by ELISA analysis. IFN-y, IL-4 and IL-2 secreted from Vα24 ⁺ iNKT cells were detected using Beadlyte® human cytokine kit (Millipore, NY, USA) and the Luminex® 200 ™ Inspection System.

Computer modeling and stimulation

The crystal structures of both human and mouse CD1d (hCD1d and mCD1d), which provide? -GalCer in each of these iNKT TCRs, have been reported in the RCSB Protein Data Bank (www.rcsb.org; PDB coded 3HUJ, 3QUX, 3QUY, and 3HE6). These crystal structures were superimposed with reference to the skeletal atoms of 3QUX. The two? -GalCer ternary structures obtained from 3HE6 and 3HUJ were used to generate other ternary complexes of different GSLs. The three-dimensional structure containing C34 was derived from containing C1 by modifying the acyl chain on Meastro (Schrodinger LLC, USA). The tertiary structure containing C1-Glc and C34-Glc was generated by inverting the O4 chirality of C1 and C34, respectively. Modeling for the remaining new terrestrial complexes was generated using these GSL and CD1d-iNKT TCR constructs from 3QUX and 3HUJ for mouse and human, respectively. All structures were processed using Protein Manufacturing Wizard (Schrodinger LLC, USA) and further abraded with the lipid tail to minimize steric clashes using the default method and OPL2005 force field in water solvent using macro model study and energy minimization Respectively. The binding of GSL to the iNKT TCR was recalculated by Autodock 4.2 using a semi-experimental free energy force field to evaluate morphology during stimulation. The estimated free energy of binding in solvent was calculated by the equation generated in Autodock 4.2:

△ G = △ V ^L (bound-unbound) + △ V ^P (bound-unbound) + △ V ^PL (bound-unbound + △ S _conf)

Wherein L refers to "ligand &quot;; P refers to "protein &quot;; V refers to pair-wise energetic terms including equations for dispersion / repulsion, hydrogen bonding, and static electricity; ΔS _conf is the evaluation of morphologic entropy lost in binding. All required inputs for performing Autodock4 were fabricated on MGLTools. 60 × 60 × 60 Å ³ grid boxes and various atomic-type energy maps. In each molecular docking run, all hydroxyl groups were set to rotate freely and the two lipid tails were assigned to their previous abraded postures. The Lamarckian genetic algorithm (5.0 x 10 ⁷ energy estimate and maximum number of 27,000 generations and a crossover rate of 0.8 and a mutation rate of 0.02) were used for the study. The results were visualized with MGLTool and the contribution of hydrogen bonding to individual residues was obtained as a built-in function. Graphic offerings finished on Maestro.

result

Glycoprotein high-grade (GSL) with αGIc is a stronger immunomodulator than those with αGal in humans, but weak in mice

Previously, 0.1 μg / mouse of 7DW8-5 was sufficient for immunostimulation, but a higher dose (1 μg / mouse) was required for the ¹⁹ glucose analog 7DW8-5-Glc to elicit an immune response (FIG. 6). Thus, the biologic activity of the newly synthesized glycolipids was tested in B6 mice at 2 and 18 hours after intravenous injection of 1 [mu] g / mouse of glycolipid. As shown in Figures 2 and 7, the Mannos analog 7DW8-5-Man failed to induce any cytokine / chemokine. Compared to GSL with αGlc, GSL with αGal has higher levels of cytokines and chemokines, including IFN-γ, IL-2, IL-4, IL-6, GM-CSF, TNFα and IP- Respectively. Similar trends have been noted for alpha GalCer and C34 analogs with different glycosyl groups (Figures 2a, 2b and 7).

Although C1-Glc and C34-Glc induced both cytokines and chemokines, 7DW8-5-Glc induced very low levels of Th1 and Th2 cytokines, but relatively high levels of KC, MCP-1, IP-10 and MIG chemokines Respectively. To detect the possibility that other immune cells other than iNKT cells could contribute to chemokine induction by 7DW8-5-Glc, JA18 KO mice without iNKT cells were treated with 7DW8-5-Glc or 7DW8 -5. Mouse sera collected at 2 hours and 18 hours after injection of ²⁵ did not show the induction of cytokines by 7DW8-5 or 7DW8-5-Glc (FIGS. 2a, 2b and 7). Surprisingly, 7DW8-5-Glc induced secretion of several chemokines including KC, MCP-1, IP-10 and MIG in Jα18 KO mice, but 7DW8-5 did not. These findings suggest that immune cells other than iNKT cells in Jα18 KO mice should contribute to the generation of chemokines in WT mice and Jα18 KO mice treated with 7DW8-5-Glc.

Then, the present inventors analyzed the expansion / activation of immune cells in WT mice on the third day after the glycolipid stimulation. The number of total T cells, CD4 ⁺ T and CD8 ⁺ T cells was higher than in mice treated with GSL with αGal heads than those treated with GSL with αGlc (FIGS. 2c, 8e and 8f). This is consistent with the observation that cytokines / chemokines in mice are induced by GSL with alpha Gal rather than GSL with alpha Glc (Figures 2a, 2b and 7). Further comparisons of immunostimulatory activity in GSL with αGlc suggest that C1-Glc and C34-Glc are responsible for the 7DW8-Cc-Glc in the induction of cytokines / chemokines (FIGS. 2a, 2b and 7) 5-Glc. &Lt; / RTI &gt; C34-Glc activated approximately two-fold more CD80 ⁺ or CD86 ⁺ DC than 7DW8-5-Glc (Figures 8c and 8d), but they induced a similar number of total spleen cells and DCs (Figures 8a and 8b). As compared to 7DW8-5-Glc, Cl-Glc not only extends about 1.3-fold more spleen cells and DC (Figures 8a and 8b), but also activates CD80 ⁺ DC about 3.5-fold and about 3-fold more CD86 ⁺ DC were activated (Figs. 8C and 8D). Increased expansion / activation of DC can contribute to stronger immunogenicity induced by C1-Glc and C34-Glc as compared to 7DW8-5-Glc in vivo. In contrast, 7DW8-5-Man did not expand any type of immune cells (Figures 2c and 8) and is consistent with the inducing members of cytokines / chemokines (Figures 2a, 2b and 7).

Unexpectedly, the present inventors also observed that 7DW8-5-Glc induced chemokines in Jα18 KO mice (FIG. 7). FACS analysis of Jα18 KO mouse splenocytes on day 3 after intravenous injection of 7DW8-5-Glc or 7DW8-5 revealed that CD11c ^hi monocyte-derived DC was significantly expanded and activated by 7DW8-5-Glc, but on 7DW8-5 (Fig. 9b-9d). No significant differences in total splenocytes, CD4 ⁺ T and CD8 ⁺ T cells were noted after stimulation with 7DW8-5 or 7DW8-5-Glc (Fig. 9a, 9e and 9f). These findings indicated that monocytes may be involved in the induction of chemokines such as KC, MCP-1, IP-10 and MIG in Jα18 KO mice treated with 7DW8-5-Glc.

As noted above, glycolipids with αGal were stronger immunomodulators than those with αGlc in mice, especially for comparison between 7DW8-5 and 7DW8-5-Glc. To investigate whether a similar trend was also applied to human iNKT cells, Vα24 ⁺ iNKT cells isolated from peripheral blood were cultured on day 2 with nonmature DC pulsed with the designated glycolipid at 100 ng / ml. After antigen removal on day 3, human iNKT cells were incubated in the presence of IL-2. The number of expanded iNKT cells was counted on the 9th day using the Guava ViaCount reagent. Surprisingly, 7DW8-5-Glc was significantly better (p = 0.0009) than 7DW8-5 in expanding human iNKT cells in vitro (Figure 2d). Taken together, these findings suggest that the biological activity of GSL with αGal is stronger in mice than in GSLs with αGlc, but less potent in humans.

mCD1d Wow Between glycolipids  Binary interaction

To understand the criteria for the difference in the immunoregulatory activity of 7DW8-5 and 7DW8-5-Glc, the present inventors used a L363 mAb capable of binding to mCD1d complexed with glycolipids, The bond strength of the interaction was measured. ²⁶ Fixed ratios of various concentrations of the mCD1d ^di -glycolipid complex were incubated with a saturating amount of L363-biotin antibody followed by streptavidin-HRP detection and ELISA (FIG. 10A). The dissociation constant (KD) between L363 and the designated mCDld ^di -glycolipid complex was calculated from the linear regression analysis of the Scatterard transformation of the plot using GraphPad prism software (Fig. 10a). L363 has been shown to recognize the mCDld -7DW8-5 ^di-Glc composite to bond strength similar to mCDld ^di -7DW8-5 complex (Fig. 10b). Next, the present inventors incubated different concentrations of glycolipids at different concentrations with a fixed amount of mCD1d dimer and L363-biotin antibody and determined the KD of the binary complex by streptavidin-HRP detection and ELISA determination (FIG. 10c). The KD was calculated from the Scatchard transformation of the binding curve derived from the L363 detection readout (Fig. 10d). Not surprisingly, 7DW8-5-Glc with the same lipid tail as 7DW8-5 bound to the mCD1d dimer with similar intensity, but their binding was about 20-fold higher than that of αGalCer. This indicated that the intensity of the binary interaction could not account for the differential immune activation capacity between 7DW8-5 and 7DW8-5-Glc.

CD1d - GSL  Complex iNKT  Intercellular Samwon  Interaction

Next, the present inventors measured the tertiary interaction between the CD1d-glycolipid complex and the iNKT TCR in mice and humans. Different concentrations of mCDld ^di -glycolipids and hCDld ^di -glycolipids complexes were incubated with fixed amounts of DN3A4-1.2 rat iNKT hybridoma cells and human Vα24 ⁺ / Vβ11 ⁺ iNKT cells, respectively. Levels of bound complexes at the indicated concentrations were detected by anti-mIgGl secondary antibody and analyzed by flow cytometry (Figs. 3a and 3b). The KD of the terahertz complex was calculated from the scatched transformation of the plot in Figures 3a and 3b using GraphPad prism software. As shown in Figure 3c, the mCD1d-7DW8-5 complex showed a stronger interaction with the iNKT TCR than the mCD1d-7DW8-5-Glc complex, about 10-fold. This was consistent with the observation that higher percent of C1-pulsed splenocytes stained by the mCDld-7DW8-5 complex (36.2 ± 5.0%) than the mCD1d-7DW8-5-Glc complex (17.1 ± 0.8% ). Both C1 (KD: 1.240 + 0.003 nM) and C34 (KD: 0.735 + 0.010 nM), when complexed with mCD1d, were C1-Glc (KD: 5.137 + 0.110 nM) and C34- nM) than the iNKT TCR (Fig. 3C).

In humans, the GSL with αGlc (KD: 8.550 ± 0.617; C34-GIc: 0.378 ± 0.019; 7DW8-5-GIc: 0.481 ± 0.008 nM of C1-Glc) / V? 11 ⁺ iNKT TCR (Figure 3D) than the V? 24 ⁺ / V? 11 ⁺ iNKT TCRs compared to the 16.410 ± 4.200; C34: 0.498 ± 0.005; 7DW8-5: 0.777 ± 0.022 nM. Thus, regardless of the lipid tail type, GSL with αGal showed a stronger triad interaction with mouse iNKT TCR than GSL with αGlc, but showed weaker triad interaction with human iNKT TCR (FIGS. 3c and 3d). This can explain the observation that GSL with αGal caused a larger expansion of the cytokines / chemokines and immune cells at higher levels in mice (FIGS. 2A-2C, 7 and 8) but resulted in less iNKT cell expansion in humans (Fig. 2D). Taken together, the triple interaction between iNKT TCR, CD1d and GSL appears to be more appropriate for the biological activity of glycolipids than the binary interaction between CD1d and GSL.

iNKT  Man vs. Mouse for Cells CD1d  Molecule Swapping  effect

To illustrate species-specific responses, the present inventors investigated the effect of swapping human-to-mouse CD1d molecules on human-to-mouse iNKT cells for the stimulatory activity of GSL with different glycosyl groups. Rat iNKT hybridoma cells (Figure 4a) or C1-expanded human Vα24 ⁺ iNKT cells (Figure 4b) were pulsed with the designated glycolipids provided by mCD1d (A20-CD1d) or hCD1d (HeLa-CD1d). The supernatant was collected after 24 hours to determine cytokine secretion. Regardless of whether it was provided by mCD1d or hCD1d, GSL with αGal induced more IL-2 secretion than GSL with αGlc in rat iNKT cell origin (FIG. 4a). This was consistent with in vivo findings that GSL with alpha Gal is stronger than GSL with alpha Glc to induce serum cytokine secretion (Figures 2 and 7). In contrast, when provided by mCD1d or hCD1d, GSL with αGlc induced more IL-2 secretion than GSL with αGal of human iNKT cell origin (FIG. 4b). Similar trends were also observed for IFN-y and IL-4 secretion from human iNKT cell origin (Figs. 12A and 12B). The species-specific stimulatory activity of GSL with different glycosyl groups was indicated by the mouse-to-human iNKT TCR rather than by CD1d. In contrast, 7DW8-5-Man was unable to stimulate mouse and human iNKT cells to secrete any cytokines regardless of their delivery by mCD1d or hCD1d. Notably, all GSL with αGlc induced significantly more Th1-biased responses than C1, based on the ratio of IFN-γ to IL-4 (FIG. 12c). Furthermore, regardless of the lipid tail, GSL with αGlc appears to be more Th1 biased than GSL with αGal in humans (FIG. 12c). These findings indicated that modification of the 4'-OH of the glycosyl group could selectively induce the response of human iNKT cells towards the Th1 direction.

CD1d - GSL - iNKT TCR of Samwon  Structural modeling of composites

To further illustrate the differential binding activity of the 3-membered complexes in mice and humans, computer modeling was performed based on the x-ray structure of the murine and human CDld-αGalCer-iNKT TCR complex, respectively (PDB accession code 3HUJ, 3QUX, 3QUY, 3QUZ, and 3HE6). ^27-29

(1) the interaction of head groups per

As shown in Figures 3a and 3b, GSL with Man could not bind to mouse and human iNKT cells when complexed with CD1d. The vertical 2'OH in mannos produced steric hindrance (iNKT Ser30 and murine Asn30 in humans) to the iNKT TCR and two hydrogen bonds originally formed by the 2'OH of galactose towards the iNKT TCR (Gly96 and Asp151 in humans and Gly96 and Asp153). For GSL with Gal, the binding of C1 to CD1d and iNKT TCR for mouse and human is shown in Figures 5a and 5b, respectively. The formation of hydrogen bonding (H-bond) interactions is most conserved, including human Asp80 (murine Asp80), human Thrl54 (mouse Thrl56), human Aspl51 (mouse Asp 153) and human Gly96 of mouse iNKT TCR Lt; / RTI &gt; On the other hand, the H-bond interactions of 3'OH / 4'OH of C1 with human and mouse iNKT TCR were very different. The residue Asn30 of mouse iNKT TCR was important for binding to the 3'- and 4'-OH of C1. The free energy contribution of Asn30 was estimated to be -2.27 ~ -3.38 Kcal / mol using MGLTools. In contrast, Ser30 of the human iNKT TCR is more remote from the 4'-OH of C1 and induces a weaker H-bond interaction with 3'-OH, but the H-bond is the 4'-OH of C1 and the skeleton of Phe29 = O groups (Fig. 5B). The free energy contributions of 3'-OH and Ser30 of C1 and 4'-OH and Phe29 of C1 were calculated to be about -1.23 to -1.63 Kcal / mol. Thus, a change of the 4'OH from the axis (Gal) to the equator (Glc) results in the loss of H-binding interactions of mouse Asn30 with human Phe29 in the iNKT TCR. As compared to C1 and C34 respectively, C1-Glc and C34-Glc lacked H-bond interactions with murine Asn30, indicating a reduced free energy (-0.7 to -0.9 Kcal / mol calculated by MGLTools) . This was consistent with a decrease in the rat three-way interaction in KD measurements when galactose was changed to the glucose head (Figure 3c). Conversely, human triad interactions were greater for GSL with glucose (Fig. 3d). Based on computer modeling, the present inventors have found that the 4 &apos; -OH of the equatorial plane of glucose is more strongly associated with the number of crystals (about -1.84 Kcal / mol) captured by human iNKT TCR-Phe51 and hCD1d-Trp153 (-0.4 Kcal / mol) of Phe29 interaction (Fig. 5C). Without the formation of hydrophobic spaces and entrapped molecules in mice, the three-way interaction is weaker for GSL with Glc than those with Gal.

(2) Interaction of lipid tail

In the acyl tail of C34, the two aromatic rings can form aromatic interactions with Phe70 and Trp63 of CD1d. Thus, a change in the truncation tail from C1 to C34 can increase the interaction with CD1d (-1.8 to -2.4 Kcal / mol by modeling). In addition, higher energy from aromatic interactions can drive the acyl chain of C34 or C34-Glc to the lower position (near Cys12) of the A 'channel in CD1d leading to subtle fluctuations in orientation of the head group (Fig. 5D). Thus, the binding of mCD1d-C34-Glc to the iNKT TCR was slightly weaker than that of mCD1d-C1-Glc (Fig. 3c), without homogeneous force between the mouse iNKT TCR and the equatorial plane 4'OH of the glucose head. This can explain why C34-Glc is less potent than C1-Glc and C34 is superior to C1 in mice.

(3) Calculated free energy using AUTODOCK4

The free energy of each GSL bound to human and mouse CD1d-iNKT TCRs was calculated three times. In each round, GSL docked human and mouse CD1d-C1-iNKT TCRs and selected the highest ranked free energy. As shown in FIG. 5E, the calculated free energy correlated with the trend of the KD value generally measured for mouse (FIG. 3c) and human (FIG. 3d).

The composition of the present invention can be formulated using various means. Techniques for formulation and administration can be found in Remington: The Science and Practice of Pharmacy, "Twentieth Edition, Lippincott Williams & Wilkins, Philadelphia, PA (1995). For human or animal administration, the formulation must meet sterility, pyrogenicity and general stability and purity standards comparable to those required by the FDA. Administration of the pharmaceutical formulations may be carried out in a variety of ways as described herein.

Cytokine induction and three-way interaction in GSL, CD1d and iNKT TCR

As compared to αGalCer, αGlcCer was reported to be less irritant to mouse iNKT cell proliferation, but was found to be more potent in stimulating human iNKT cell proliferation. ^{5-22 In} our study, the differential activity between alphaCalCer and alpha GlcCer in mice and humans was also observed for cytokine induction and three-way interactions in GSL, CD1d, and iNKT TCR. In addition, phenyl GSL containing αGal or αGlc head showed similar species-specific activity. Thus, regardless of the lipid tail, GSL with αGal was superior to GSL with αGlc in mice, but was worse in humans. This indicated that although the CD1d and iNKT TCRs are highly homologous between mouse and human, the biological activity of glycolipids in mice can not be interpreted as biological activity in humans. ³⁰

The species-specific response is likely to be due to differences in the iNKT TCR between mouse and human, as evidenced by mCD1d versus hCD1d swapping analysis. GSL with αGal provided by mCD1d or hCD1d was stronger than GSL with αGlc in stimulating rat iNKT cells but less stimulating for human iNKT cells. According to the crystal structure of CD1d-αGalCer-iNKT TCR, the residues in contact with the 4'-OH of the galactose head in the CDR1α region of the iNKT TCR were not conserved between mouse (Asn30) and human (Phe29). ^27,28 Computer modeling has shown that the change in 4'-OH from the axis to the equatorial plane loses hydrogen bonding between 4'OH and iNKT TCR-Asn30 in mice. Conversely, the equatorial plane 4'-OH of glucose was found to be able to compensate for the loss of Phe29 interaction by stronger interactions of the crystal number, captured by human iNKT TCR-Phe51 and hCD1d-Trp153. Thus, the three-way interaction composed of GSL with αGlc was stronger than GSL with αGal in humans, but weak in mice. Taken together, structural modeling of murine and human CD1d-GSL-iNKT TCR complexes provided excellent clarification for species-specific biological activity and the calculated free energy change correlated well with the trend of measured binding of tertiary complexes .

In contrast to our binary-forming analogues with the same tail but with different glycosyl groups, the species-specific immune response also appeared in other cases with the same glycosyl group but with different lipid tail. ^{31 and 32.} Similarly, this was CD1d instead of iNKT TCR which form two first activity of the C- glycoside analogs between bond strength and well-correlated to mouse and human of the three-way complex. ^{31 For} the glycolipids of this invention, the three-way interaction was also more important than the binary interaction in predicting the biological response in mice and humans. Both 7DW8-5 and 7DW8-5-Glc bound to mCD1d more strongly than C1, presumably due to increased contact between the phenyl ring and the fluorine atom and the CD1d A 'pocket. ³³ Two glycolipids with the same lipid tail exhibited similar binding strengths to CD1d, but they showed different immunostimulatory effects. 7DW8-5-Glc was superior to 7DW8-5 in humans, but was poor in mice, which had a good relationship with the binding of the tertiary complex. In addition to 7DW8-5 and 7DW8-5-Glc, the biological activity of the other two pairing analogues (C1 versus C1-Glc and C34 versus C34-Glc) is also well correlated with the intensity of the three- . That is, GSL with αGal showed stronger three-way interaction and immunostimulatory activity than GSL with αGlc in mouse, but this tendency was reversed in humans. Thus, measurement of in vitro three-way interactions can be used to predict the immunostimulatory potency of new glycolipids in vivo.

Further comparisons among GSL with αGlc revealed that both C1-Glc and C34-Glc are superior to 7DW8-5-Glc in cytokine induction in mice. However, the binding of the murine terpenoid complex was comparable to that of C34-Glc and 7DW8-5-Glc. These findings suggest that other factors besides KD can also modulate the in vivo immune response. In fact, both C1-Glc and C34-Glc activated more CD80 ⁺ or CD86 ⁺ DC than 7DW8-5-Glc, which could contribute to the stronger biological activity of C1-Glc and C34-Glc in mice. Taken together, several factors can regulate in vivo immune stimulation, including the three-way interaction and expansion / activation intensity of immune cells.

In contrast to GSL with αGal or αGlc, αManCer and 7DW8-5-Man failed to induce expansion / activation of iNKT cell proliferation ^{5 , 22} , cytokine / chemokine and / or immune cells in both mouse and human . This may be due to the fact that the rat or human iNKT TCR can not recognize the αMan head as evidenced by the absence of staining of the CD1d-7DW8-5-Man dimer in iNKT cells. As compared to 7DW8-5-Man, 7DW8-5-Glc was able to induce Th1 and Th2 cytokines, albeit at a very low level in mice. Relatively, large amounts of KC, MCP-1, IP-10 and MIG chemokines were induced by 7DW8-5-Glc in WT and Ja18 KO mice, suggesting that certain other types of immune cells other than iNKT cells contribute to these chemokine secretions . Indeed, monocytes were significantly expanded / activated in Jα18 KO mice by 7DW8-5-Glc. Monocytes have been reported to be capable of producing these chemokines in response to stimuli and ^34-37 suggesting that monocytes may be involved in chemokine secretion in 7DW8-5-Glc-treated mice. Nevertheless, the present inventors could not rule out that other possible sources present in Jα18 KO mice could also play a role. Vα1O NKT cells were able to produce IFN-γ and IL-4 in vitro in response to αGalCer and αGlcCer, but IFN-γ was not secreted in the serum of Jα18 KO mice treated with αGalCer. ^{39 The} present inventors could not detect any cytokine production from 7DW8-5 or 7DW8-5-Glc treated Ja18 KO mice. These findings imply that Vα10 NKT cells contributed little to chemokines induced by 7DW8-5-Glc in mice.

However, most cytokines and chemokines, including IFN-y, IL-2, IL-4, IL-6, GM-CSF and TNFa are not produced in JA18 KO mice stimulated with 7DW8-5 or 7DW8-5-Glc I did. Immune cells such as CD4 ⁺ T and CD8 ⁺ T cells did not expand in Jα18 KO mice. Thus, iNKT cells remain a major player in the above-mentioned cytokine / chemokine induction and immune cell expansion by 7DW8-5 or 7DW8-5-Glc.

In summary, GSL with αGlc possessed stronger three-dimensional interactions and induced more Th1-biased immunity in comparison to GSL with αGal in humans. However, GSL with alpha Glc was less irritating than GSL with alpha Gal in mice. The species-specific response was due to the differential binding of species-terrestrial complexes, reflecting the difference between mouse and human iNKT TCR as supported by mCD1d versus hCD1d swapping analysis. This was consistent with computer modeling predictions based on the crystal structure of the CDld-αGalCer-iNKT TCR complex in mice and humans. ^{27-29 In} addition to the three-way interaction between the CD1d-glycolipid complex and the iNKT TCR, extended / activated mononuclear cells are also capable of modulating the in vivo immune response to GSL with αGlc in particular.

From this study, changes in the 4'OH direction on glycosyl groups induced different biological activities in mice and humans. This is consistent with reports that although the aromatic group introduced into the 4'OH of the αGalCer head may affect the production of iNKT cell cytokines in mice, their effect in humans has not been investigated. ⁴⁰ Changes in glycosyl groups at position 6 also showed various effects on biological responses. ^29,41 These findings together with the present study provide a new direction for the future design and synthesis of the new GSL.

Throughout this application, various publications, patents, and published patent applications are cited. These publications, patents, and published patent applications referred to herein are incorporated herein by reference in their entirety. The citation of publications, patents, or published patent publications herein does not imply that the publications, patents, or published patent applications are prior art.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent source were specifically and individually indicated to be incorporated herein by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is to be understood that certain changes and modifications may be effected therein without departing from the spirit or scope of the appended claims, Will be readily apparent to those skilled in the art.

Claims (48)

An immunoadjuvant compound having the formula (I) or a pharmaceutically acceptable salt thereof.
Formula I

In this formula,
R ¹ is -OH or halogen;
R ² is -OH, hydrogen or halogen;
R &lt; ³ &gt; is hydrogen or halogen;
Each occurrence of R ⁴ and R ⁵ is independently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocycle Optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted amino group, or optionally substituted acyl;
n is an integer from 1 to 15;
and m is an integer of 1 to 20. 2. The compound of claim 1, wherein R &lt; ² &gt; is -OH. 2. The compound of claim 1, wherein R &lt; ² &gt; is halogen. 4. The compound according to any one of claims 1 to 3, wherein R &lt; ¹ &gt; is -OH. 4. The compound according to any one of claims 1 to 3, wherein R &lt; ¹ &gt; is halogen. 3. The compound of claim 1, wherein R &lt; ⁴ &gt;
(II)

In this formula,
i is 0, 1, 2, 3, 4, or 5;
R ⁶ is independently hydrogen, _{halogen, -CN, -NO 2, ~ N} 3, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally Optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted amino group, or optionally substituted acyl. 7. A compound according to claim 6 wherein R &lt; ⁶ &gt; is halogen. 7. Compounds according to claim 6, wherein R &lt; ⁶ &gt; 2. The compound according to claim 1, wherein R &lt; ⁴ &gt;

In this formula,
j is 0, 1, 2, 3, or 4;
k is 0, 1, 2, 3, 4, or 5;
Each occurrence of R ⁷ and R ⁸ is independently hydrogen, halogen, -CN, -NO ₂ , -N ₃ , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl , An optionally substituted aryl, an optionally substituted heterocyclyl, an optionally substituted heteroaryl, an optionally substituted alkoxy, an optionally substituted amino group, or an optionally substituted acyl. ^{10. The} compound of claim 9, wherein each occurrence of R &lt; ⁷ &gt; and R &lt; ⁸ &gt; is independently hydrogen or halogen. ^10. The compound of claim 9, wherein R &lt; ⁷ &gt; is hydrogen; R &lt; ⁸ &gt; is F; and k is 1, 2 or 3. ^10. The compound of claim 9, wherein R &lt; ⁷ &gt; is F; R &lt; ⁸ &gt; is hydrogen; and j is 1, 2 or 3. ^{10. The} compound of claim 9, wherein R &lt; ⁷ &gt; and R &lt; ⁸ &gt; are both F; k is 1, 2 or 3; and j is 1, 2 or 3. 2. The compound of claim 1, wherein said compound is selected from one of the following compounds.

(i) a compound according to any one of claims 1 to 14 in an amount sufficient to stimulate an immune response when co-administered with a human subject in combination with an antigen, and (ii) a pharmaceutically acceptable excipient A pharmaceutical composition. Comprising co-administering the antigen together with an adjuvant composition comprising a GSL compound of formula I, as a method for increasing the immunogenicity of an antigen in a subject in need thereof. 15. A method for stimulating an immune response in a human subject in need thereof comprising administering to a subject a therapeutically effective amount of an immunoadjuvant composition in a pharmaceutically acceptable carrier, Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; 17. The method of claim 16, wherein the adjuvant composition is a vaccine adjuvant. 17. The method of claim 16, wherein the adjuvant composition is administered in an amount that is capable of elevating an invariant Natural Killer T (iNKT) cell in a human. 20. The method of claim 19, wherein administration of the adjuvant composition increases cytokine and / or chemokine production in a human. 21. The method of claim 20, wherein said cytokine production is sufficient to transactivate downstream immune cells. 22. The method of claim 21, wherein said downstream immune cells comprise at least one dendritic cell (DC), natural killer cell (NK), B cell, CD4 ⁺ T and CD8 ⁺ T cells. 21. The method of claim 20, wherein the cytokine comprises a Th1 cytokine. 24. The method of claim 23, wherein said Th1 cytokine is selected from at least one of the group consisting of interferon-gamma (IFN-?), GM-CSF, TNF ?, interleukin 2, interleukin 12 and interleukin 10. 21. The method of claim 20, wherein the chemokine is selected from at least one of the group comprising RANTES, MIP-1 alpha, KC, MCP-I, IP-10 and MIG. 17. The method of claim 16, wherein administration of the composition has an anti-cancer effect. 27. The method of claim 26, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, leukemia, solid tumors and carcinomas. 17. Compounds of formula I according to claim 16, wherein in compounds of formula I R &lt; ⁴ &gt; is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, wherein the increase in Th1 cytokine in a human exceeds a certain increase in Th2 cytokines . 1. A method of increasing invasive natural killer T (iNKT) cell production in a human subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition, &Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof. 30. The method of claim 29 wherein the elevation of the level of iNKT is greater than when compared to an elevation resulting from the administration of an equivalent amount of a glyceral analog that comprises alpha-galactose (alpha Gal) as a glycosyl head group. A method for stimulating the production of cytokines and / or chemokines in a human subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition, wherein the composition is effective for increasing cytokine / chemokine production Comprising a compound according to any one of claims 1 to 14 in a sufficient amount. 32. The method of claim 31, wherein said cytokine production is sufficient to transactivate downstream immune cells. 33. The method of claim 32, wherein said downstream immune cells comprise at least one of dendritic cells (DC), natural killer cells (NK), B cells, CD4 ⁺ T and CD8 ⁺ T cells. 32. The method of claim 31, wherein said cytokine comprises a Th1 cytokine. 35. The method of claim 34, wherein the cytokine is selected from interferon-gamma (IFN-y), GM-CSF, TNFa, interleukin 2, interleukin 12 and interleukin 10. 32. The method of claim 31, wherein the chemokine is selected from RANTES, MIP-1 alpha, KC, MCP-I, IP-10, and MIG. 16. The pharmaceutical composition according to claim 15, wherein said composition is a vaccine adjuvant. 16. The pharmaceutical composition according to claim 15, wherein the composition is an anticancer therapeutic. 17. A pharmaceutical composition according to claim 16, wherein R &lt; ⁴ &gt; in the compound is selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, said compound being capable of increasing Thl cytokines with a minimal involvement of Th2 cytokines . 31. A method for enhancing an immune response by an antigen in a subject comprising administering to the subject an effective amount of a vaccine comprising at least one antigen and an immunologically effective amount of an adjuvant composition of claim 15. 41. The method of claim 40, wherein the one or more antigens are selected from the group consisting of bacterial antigens, viral antigens, fungal antigens, protozoal antigens, prion antigens, neo antigens, tumor antigens and auto-antigens. 41. The method of claim 40, wherein the vaccine comprises a nucleic acid, a protein, a peptide, a glycoprotein, a carbohydrate, a fusion protein, a lipid, a glycolipid, a carbohydrate-protein conjugate; Cell or extract thereof; Dead or attenuated cells or extracts thereof; Tumor cells or extracts thereof; Virus particles; Allergen, or a mixture thereof. 41. The method of claim 40, wherein the administered antigen is a tumor antigen. 41. The method of claim 40, wherein the amount of antigen is administered in the range of 0.1 μg to 100 mg per kg of body weight. 41. The method of claim 40, wherein the amount of adjuvant is in the range of 10 to 100 micrograms per kilogram of body weight. 41. The method of claim 40, wherein said co-administered composition is a co-formulated pharmaceutically acceptable composition comprising a GSL of formula I and a pharmaceutically acceptable carrier. A product comprising GSL of formula (I). A kit comprising a GSL according to any one of claims 1 to 14 and instructions for use.

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